Alpha-1 AntiTrypsin (AAT) RNAi Agents, Compositions Including AAT RNAi Agents, And Methods of Use

ABSTRACT

RNAi agents for inhibiting the expression of the alpha-1 antitrypsin (AAT) gene, compositions including AAT RNAi agents, and methods of use are described. Also disclosed are pharmaceutical compositions including one or more AAT RNAi agents together with one or more excipients capable of delivering the RNAi agent(s) to a liver cell in vivo. Delivery of the AAT RNAi agent(s) to liver cells in vivo inhibits AAT gene expression and treats diseases associated with AAT deficiency such as chronic hepatitis, cirrhosis, hepatocellular carcinoma, transaminitis, cholestasis, fibrosis, and fulminant hepatic failure.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/867,107, filed on Jan. 10, 2018, and claims priority to U.S.Provisional Patent Application Ser. No. 62/596,232, filed on Dec. 8,2017, U.S. Provisional Patent Application Ser. No. 62/486,720, filed onApr. 18, 2017, and U.S. Provisional Patent Application Ser. No.62/444,452, filed on Jan. 10, 2017, the contents of each of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

Disclosed herein are RNA interference (RNAi) agents for inhibition ofalpha-1 antitrypsin gene expression, compositions that include alpha-1antitrypsin RNAi agents, and methods of use thereof.

BACKGROUND

Alpha-1 antitrypsin (AAT, al-antitrypsin, or A1AT) deficiency is aninherited, autosomal codominant genetic disorder that causes misfoldingof the AAT protein and poor secretion of the misfolded protein leadingto lung and liver diseases. AAT deficiency (AATD) occurs with afrequency of about 1 in every 1,500 to 3,500 individuals and most oftenaffects persons with European ancestry.

Alpha-1 Antitrypsin is a protease inhibitor belonging to the serpinsuperfamily. Normal AAT protein is a circulating glycoprotein proteaseinhibitor primarily synthesized in the liver by hepatocytes and secretedinto the blood. The known physiologic function of AAT is to inhibitneutrophil proteases, which serves to protect host tissues fromnon-specific injury during periods of inflammation.

The most clinically significant form of AATD, a genetic disorderassociated with liver disease in children and adults, and pulmonarydisease in adults, is caused by the Z mutation. The Z mutant allele(PiZ), through a single point mutation, renders the mutant Z form AATprotein (the “Z-AAT protein”) prone to abnormal folding causingintracellular retention. The mutant Z-AAT protein monomers are able toform chains of polymers that amass into aggregates, which are sometimesreferred to as “globules.” The misfolded Z-AAT protein is ineffective intraversing the secretory pathway, and instead polymerizes andaccumulates in the endoplasmic reticulum (ER) of hepatocytes. Thepolymeric globule masses stress the ER and trigger continuous hepatocyteinjury, leading to fibrosis, cirrhosis, and increased risk ofhepatocellular carcinoma. Further, the absence of circulatinganti-protease activity leaves the lung vulnerable to injury byneutrophil elastase, resulting in the development of respiratorycomplications such as emphysema.

Individuals with the homozygous PiZZ genotype have severe deficiency offunctional AAT, which leads to pulmonary disease. Weekly use of AATaugmentation therapy, using purified human AAT, results in near normalplasma levels of AAT in subjects with AATD, and helps prevent lungdamage in affected individuals. However, while the administration ofpurified AAT can ameliorate or help prevent lung damage caused by theabsence of endogenously secreted AAT, AATD patients remain vulnerable toendoplasmic reticulum liver storage disease caused by the deposition andaccumulation of excessive abnormally folded AAT protein. AccumulatedZ-AAT protein in the globule conformation in hepatocytes is a well-knowncharacteristic of AATD liver disease and is believed to lead toproteotoxic effects that are responsible for inducing liver injury,including liver cell damage and death and chronic liver injury, inindividuals with AATD. (see, e.g., D. Lindblad et al., Hepatology 2007,46: 1228-1235). Patients with AATD often develop liver disease, whichcan be severe or fatal, even in infancy. Clinical presentations ofinjury in the liver include chronic hepatitis, cirrhosis, hepatocellularcarcinoma, transaminitis, cholestasis, fibrosis, and even fulminanthepatic failure.

There is currently no clinically approved treatment to prevent the onsetor slow the progression of liver disease due to AATD. Further, whileU.S. Patent Application Publication No. 2015/0361427 discloses certainRNAi agents capable of inhibiting the expression of an AAT gene, thereremains a need for novel and effective AAT RNAi agents having improvedpotency that can selectively, efficiently, and safely inhibit theexpression of an AAT gene, thereby preventing and potentially reversingZ-AAT accumulation-related liver injury and fibrosis. Similarly, whileU.S. Patent Application Publication No. 2015/0011607 to Brown et al.(“Brown '607”) discloses various sequences for inhibiting expression ofan AAT gene, Brown teaches the use of longer double-stranded constructs(referred to in Brown as DsiRNAs), which according to Brown have beenfound to give “unexpected effective results in terms of potency andduration of action” as compared to 19-23mer siRNA agents. (See, e.g.,Brown '607 at paragraph [0376]). Moreover, many of the sequencesdisclosed in Brown '607 are designed to be used in DsiRNA constructsthat are designed to target different locations of an AAT mRNA ascompared to the sequences disclosed in the present invention. Suchdifferences lead to different binding affinity to the AAT mRNA andproduces a different cleavage site, which can impact the inhibitoryeffect of the compound, while also potentially leading to additionaloff-target issues (see, e.g., Piotr J. Kamola et al., PLoS Comput Biol,2015, 11(12):e1004656 at FIG. 1 (illustrating the mechanism ofsiRNA-Mediated Gene Silencing)). For example, nothing in Brown ‘607teaches or suggests the design of an RNAi agent (of any length) whereinthe 5’ terminal nucleobase or nucleotide of the antisense strand wouldbe aligned with the position that is 19 nucleotides downstream (towardsthe 3′ end) from position 1000 on an AAT gene (SEQ ID NO: 1). Putdifferent, and again solely as an example involving one such potentialAAT RNAi agent sequence, nothing in Brown ‘607 teaches or suggests thedesign of an RNAi agent wherein the 5’ terminal nucleobase of theantisense strand of an RNAi agent corresponds to position 1018 on an AATgene (SEQ ID NO: 1). Further, nothing in Brown '607 teaches or suggeststhe modified AAT RNAi agent constructs disclosed herein.

SUMMARY

There exists a need for novel AAT-specific RNA interference (RNAi)agents (also herein termed RNAi agent, RNAi trigger, or trigger) thatare able to selectively and efficiently inhibit the expression of an AATgene. Further, there exists a need for compositions of novelAAT-specific RNAi agents for the treatment of diseases associated withAAT deficiency.

Because liver damage resulting from AATD occurs through again-of-function mechanism, inhibition of AAT gene expression is usefulin preventing accumulation of the Z-AAT protein in the liver. Further,the reduction or removal of the Z-AAT polymer aggregates reduces the ERstress in hepatocytes, and offers additional advantages in reducing thelikelihood of occurrence of liver cell damage and assisting in thetreatment of liver cell damage and chronic liver injury such asfibrosis, cirrhosis, hepatocellular carcinoma, and other conditions anddiseases caused by AATD. Reduction of inflammatory Z-AAT protein, whichhas been clearly defined as the cause of progressive liver disease inAATD patients, is important as it can slow or halt the progression ofliver disease and allow fibrotic tissue repair.

In general, the disclosure features novel AAT RNAi agents, compositionscomprising the AAT RNAi agents, and methods for inhibiting theexpression of an AAT gene in vivo and/or in vitro using AAT RNAi agentsand compositions that include AAT RNAi agents. Further described hereinare methods of treatment of AATD-related diseases using the AAT RNAiagents described herein and compositions that include AAT RNAi agents.

The AAT RNAi agents and methods disclosed herein can provide for thetreatment of AATD, including the treatment of conditions and diseasescaused by AATD, such as chronic hepatitis, cirrhosis, hepatocellularcarcinoma, and fulminant hepatic failure. The AAT RNAi agents disclosedherein, when administered to a subject, can prevent and/or reverse Z-AATaccumulation-related liver injury and fibrosis. The AAT RNAi agentsdescribed herein may be administered to a subject, e.g., a human oranimal subject, by any suitable methods known in the art, such assubcutaneous injection or intravenous administration.

In one aspect, the disclosure features RNAi agents for inhibiting theexpression of an alpha-1 antitrypsin (AAT) gene, wherein the RNAi agentcomprises a sense strand and an antisense strand. Also described hereinare compositions comprising an RNAi agent capable of inhibiting theexpression of an alpha-1 antitrypsin gene, wherein the RNAi agentcomprises a sense strand and an antisense strand, and at least onepharmaceutically acceptable excipient.

Each AAT RNAi agent described herein includes a sense strand and anantisense strand. The sense strand and the antisense strand can bepartially, substantially, or fully complementary to each other. Thelength of the RNAi agent sense and antisense strands described hereineach can be 16 to 30 nucleotides in length. In some embodiments, thesense and antisense strands are independently 17 to 26 nucleotides inlength. In some embodiments, the sense and antisense strands areindependently 21 to 26 nucleotides in length. In some embodiments, thesense and antisense strands are independently 21 to 24 nucleotides inlength. In some embodiments, the sense and/or antisense strands areindependently 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleotides in length. The sense and antisense strands can be eitherthe same length or different lengths. The RNAi agents described herein,upon delivery to a cell expressing AAT, inhibit the expression of one ormore AAT genes in vivo or in vitro.

An AAT RNAi agent includes a sense strand (also referred to as apassenger strand), and an antisense strand (also referred to as a guidestrand). A sense strand of the AAT RNAi agents described herein includesa nucleotide sequence having at least 85% identity to a core stretch ofat least 16 consecutive nucleotides to a sequence in an AAT mRNA. Insome embodiments, the sense strand core stretch having at least 85%identity to a sequence in an AAT mRNA is 16, 17, 18, 19, 20, 21, 22, or23 nucleotides in length. An antisense strand of an AAT RNAi agentincludes a nucleotide sequence having at least 85% complementarity overa core stretch of at least 16 consecutive nucleotides to a sequence inan AAT mRNA and the corresponding sense strand. In some embodiments, theantisense strand core stretch having at least 85% complementarity to asequence in an AAT mRNA or the corresponding sense strand is 16, 17, 18,19, 20, 21, 22, or 23 nucleotides in length.

In some embodiments, the AAT RNAi agents disclosed herein target aportion of an AAT gene having the sequence of any of the sequencesdisclosed in Table 1.

Examples of AAT RNAi agent sense strands and antisense strands that canbe used in AAT RNAi agents are provided in Tables 2, 3, 4 and 5.Examples of duplexes that include AAT RNAi agent are provided in Table6. Examples of 19-nucleotide core stretch sequences that may consist ofor may be included in the sense strands and antisense strands of certainAAT RNAi agents disclosed herein, are provided in Table 2.

In another aspect, the disclosure features methods for delivering AATRNAi agents to liver cells in a subject, such as a mammal, in vivo. Insome embodiments, one or more AAT RNAi agents are delivered to targetcells or tissues using any oligonucleotide delivery technology known inthe art. Nucleic acid delivery methods include, but are not limited to,encapsulation in liposomes, iontophoresis, or incorporation into othervehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules,and bioadhesive microspheres, proteinaceous vectors, or DynamicPolyconjugates™ (DPCs) (see, for example WO 2000/053722, WO2008/0022309, WO 2011/104169, and WO 2012/083185, each of which isincorporated herein by reference). In some embodiments, a deliveryvehicle, such as a polymer, an amphipathic polymer, a membrane activepolymer, a peptide, such as a melittin or melittin-like peptide, areversibly modified polymer or peptide, or a lipid, can be used with theAAT RNAi agents disclosed herein.

In some embodiments, an AAT RNAi agent is delivered to target cells ortissues by covalently linking or conjugating the RNAi agent to atargeting group such as an asialoglycoprotein receptor ligand. In someembodiments, an asialoglycoprotein receptor ligand includes, consistsof, or consists essentially of, a galactose or galactose-derivativecluster. In some embodiments, an AAT RNAi agent is linked to a targetingligand comprising the galactose-derivative N-acetyl-galactosamine. Insome embodiments, a galactose-derivative cluster includes anN-acetyl-galactosamine trimer or an N-acetyl-galactosamine tetramer. Insome embodiments, a galactose derivative cluster is anN-acetyl-galactosamine trimer or an N-acetyl-galactosamine tetramer.Example targeting groups useful for delivering RNAi agents aredisclosed, for example, in U.S. patent application Ser. No. 15/452,324and WO 2017/156012, which are incorporated by reference herein in theirentirety.

A targeting group can be linked to the 3′ or 5′ end of a sense strand oran antisense strand of an AAT RNAi agent. In some embodiments, atargeting group is linked to the 3′ or 5′ end of the sense strand. Insome embodiments, a targeting group is linked to the 5′ end of the sensestrand. In some embodiments, a targeting group is linked internally to anucleotide on the sense strand and/or the antisense strand of the RNAiagent. In some embodiments, a targeting group is linked to the RNAiagent via a linker.

A targeting group, with or without a linker, can be linked to the 5′ or3′ end of any of the sense and/or antisense strands disclosed in Tables2, 3, 4, and 5. A linker, with or without a targeting group, can beattached to the 5′ or 3′ end of any of the sense and/or antisensestrands disclosed in Tables 2, 3, 4, and 5.

In another aspect, the disclosure features compositions that include oneor more AAT RNAi agents that have the duplex structures disclosed inTable 6.

In some embodiments, described herein are compositions that include acombination or cocktail of at least two AAT RNAi agents having differentnucleotide sequences. In some embodiments, the two or more different AATRNAi agents are each separately and independently linked to targetinggroups. In some embodiments, the two or more different AAT RNAi agentsare each linked to targeting groups that include or consist of targetingligands that include one or more moieties that target anasialoglycoprotein receptor. In some embodiments, the two or moredifferent AAT RNAi agents are each linked to targeting groups thatinclude or consist of targeting ligands that include one or moregalactose-derivatives. In some embodiments, the two or more differentAAT RNAi agents are each linked to targeting groups that include orconsist of targeting ligands that include one or moreN-acetyl-galactosamines. In some embodiments, when two or more RNAiagents are included in a composition, each RNAi agent is independentlylinked to the same targeting group. In some embodiments, when two ormore RNAi agents are included in a composition, each RNAi agent isindependently linked to a different targeting group, such as targetinggroups having different chemical structures.

In some embodiments, targeting groups are linked to the AAT RNAi agentswithout the use of an additional linker. In some embodiments, thetargeting group is designed having a linker readily present tofacilitate the linkage to an AAT RNAi agent. In some embodiments, whentwo or more RNAi agents are included in a composition, the two or moreRNAi agents may be linked to their respective targeting groups using thesame linkers. In some embodiments, when two or more RNAi agents areincluded in a composition, the two or more RNAi agents are linked totheir respective targeting groups using different linkers.

In another aspect, the disclosure features methods for inhibitingalpha-1 antitrypsin gene expression in a subject, the methods comprisingadministering to the subject an amount of an AAT RNAi agent capable ofinhibiting the expression of an AAT gene, wherein the AAT RNAi agentcomprises a sense strand and an antisense strand.

Also described herein are methods for the treatment of a condition ordisease caused by AATD, comprising administering to a subject atherapeutically effective amount of an RNAi agent described herein.Further described are methods for inhibiting expression of an AAT gene,wherein the methods include administering to a cell an AAT RNAi agentdescribed herein.

In some embodiments, disclosed herein are methods for the treatment ofAATD (including the treatment of a condition or disease caused by AATD),the methods comprising administering to a subject a therapeuticallyeffective amount of an RNAi agent having an antisense strand comprisingthe sequence of any of the sequences in Tables 2, 3, or 4.

In some embodiments, disclosed herein are methods for inhibitingexpression of an AAT gene, the methods comprising administering to acell an AAT RNAi agent that includes an antisense strand comprising thesequence of any of the sequences in Tables 2, 3. or 4.

In some embodiments, disclosed herein methods for the treatment of AATD(including the treatment of a condition or disease caused by AATD), themethods comprising administering to a subject a therapeuticallyeffective amount of an RNAi agent that includes a sense strandcomprising the sequence of any of the sequences in Tables 2, 3, or 5.

In some embodiments, disclosed herein are methods for inhibitingexpression of an AAT gene, wherein the methods include administering toa cell an AAT RNAi agent that includes a sense strand comprising thesequence of any of the sequences in Tables 2, 3, or 5.

In some embodiments, disclosed herein are methods for the treatment ofAATD (including the treatment of a condition or disease caused by AATD),wherein the methods include administering to a subject a therapeuticallyeffective amount of an RNAi agent that includes a sense strandcomprising the sequence of any of the sequences in Table 5, and anantisense strand comprising any of the sequences in Table 4.

In some embodiments, disclosed herein are methods for inhibitingexpression of an AAT gene, wherein the methods include administering toa subject a therapeutically effective amount of an RNAi agent thatincludes a sense strand comprising the sequence of any of the sequencesin Table 5, and an antisense strand comprising any of the sequences inTable 4.

In some embodiments, disclosed herein are methods of inhibitingexpression of an AAT gene, wherein the methods include administering toa subject an AAT RNAi agent that includes a sense strand consisting ofthe nucleobase sequence of any of the sequences in Table 5, and theantisense strand consisting of the nucleobase sequence of any of thesequences in Table 4. In other embodiments, disclosed herein are methodsof inhibiting expression of an AAT gene, wherein the methods includeadministering to a subject an AAT RNAi agent that includes a sensestrand consisting of the modified sequence of any of the modifiedsequences in Table 5, and the antisense strand consisting of themodified sequence of any of the modified sequences in Table 4.

In some embodiments, disclosed herein are methods for inhibitingexpression of an AAT gene in a cell, wherein the methods includeadministering one or more AAT RNAi agents having the duplex structureset forth in Table 6.

In some embodiments, the AAT RNAi agents disclosed herein havestructures that include, consist of, or consist essentially of, thestructure shown in any one of FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5,FIG. 6, FIG. 7, or FIG. 8.

The AAT RNAi agents disclosed herein are designed to target specificpositions on an AAT gene (SEQ ID NO:1). As defined herein, an antisensestrand sequence is designed to target an AAT gene at a given position onthe gene when the 5′ terminal nucleobase of the antisense strand wouldbe aligned with the position that is 19 nucleotides downstream (towardsthe 3′ end) from the position on the gene when base pairing to the gene.For example, as illustrated in Tables 1, 2, and 3 herein, an antisensestrand sequence designed to target an AAT gene at position 1000 requiresthat when base pairing to the gene, the 5′ terminal nucleobase of theantisense strand is aligned with position 1018 of the AAT gene. Asprovided herein, an AAT RNAi agent does not require that the nucleobaseat position 1 (5′→3′) of the antisense strand be complementary to thegene, provided that there is at least 85% complementarity (e.g., atleast 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity)of the antisense strand and the gene across a core stretch sequence ofat least 16 consecutive nucleotides. For example, for an AAT RNAi agentdisclosed herein that is designed to target position 1000 of an AATgene, the 5′ terminal nucleobase of the antisense strand of the of theAAT RNAi agent must be aligned with position 1018 of the gene; however,the 5′ terminal nucleobase of the antisense strand may be, but is notrequired to be, complementary to position 1018 of an AAT gene, providedthat there is at least 85% complementarity (e.g., at least 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100% complementarity) of the antisensestrand and the gene across a core stretch sequence of at least 16consecutive nucleotides. As shown by, among other things, the variousexamples disclosed herein, the specific site of binding of the gene bythe antisense strand of the AAT RNAi agent (e.g., whether the AAT RNAiagent is designed to target an AAT gene at position 1000, at position1142, or at some other position) is highly important to the level ofinhibition achieved by the AAT RNAi agent.

In some embodiments, the antisense strand sequence is designed to have asequence target position 1000 of an AAT gene (SEQ ID NO: 1).

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCU (SEQ IDNO: 801), wherein at least one or more nucleotides is a modifiednucleotide. In some embodiments, the antisense strand of an AAT RNAiagent comprises or consists of the nucleobase sequence ofUGUUAAACAUGCCUAAACGCU (SEQ ID NO: 801), wherein all or substantially allof the nucleotides are modified nucleotides.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGUU (SEQ IDNO: 794), wherein at least one or more nucleotides is a modifiednucleotide. In some embodiments, the antisense strand of an AAT RNAiagent comprises or consists of the nucleobase sequence ofUGUUAAACAUGCCUAAACGUU (SEQ ID NO: 794), wherein all or substantially allof the nucleotides are modified nucleotides.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCUU (SEQ IDNO: 839), wherein at least one or more nucleotides is a modifiednucleotide. In some embodiments, the antisense strand of an AAT RNAiagent comprises or consists of the nucleobase sequence ofUGUUAAACAUGCCUAAACGCUU (SEQ ID NO: 839), wherein all or substantiallyall of the nucleotides are modified nucleotides.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCG (SEQ IDNO: 800), wherein at least one or more nucleotides is a modifiednucleotide. In some embodiments, the antisense strand of an AAT RNAiagent comprises or consists of the nucleobase sequence ofUGUUAAACAUGCCUAAACGCG (SEQ ID NO: 800), wherein all or substantially allof the nucleotides are modified nucleotides.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACG (SEQ IDNO: 80), wherein one or more nucleotides is a modified nucleotide. Insome embodiments, the antisense strand of an AAT RNAi agent comprises orconsists of the nucleobase sequence of UGUUAAACAUGCCUAAACG (SEQ ID NO:80), wherein all or substantially all of the nucleotides are modifiednucleotides.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of AGUUAAACAUGCCUAAACG (SEQ IDNO: 81), wherein one or more nucleotides is a modified nucleotide. Insome embodiments, the antisense strand of an AAT RNAi agent comprises orconsists of the nucleobase sequence of AGUUAAACAUGCCUAAACG (SEQ ID NO:81), wherein all or substantially all of the nucleotides are modifiednucleotides.

In some embodiments, the sense strand of an AAT RNAi agent comprises orconsists of the nucleobase sequence of CGUUUAGGCAUGUUUAACA (SEQ ID NO:429), wherein one or more nucleotides is a modified nucleotide. In someembodiments, the sense strand of an AAT RNAi agent comprises or consistsof the nucleobase sequence of CGUUUAGGCAUGUUUAACA (SEQ ID NO: 429),wherein all or substantially all of the nucleotides are modifiednucleotides.

In some embodiments, the sense strand of an AAT RNAi agent comprises orconsists of the nucleobase sequence of CGUUUAGGCAUGUUUAACU (SEQ ID NO:430), wherein one or more nucleotides is a modified nucleotide. In someembodiments, the sense strand of an AAT

RNAi agent comprises or consists of the nucleobase sequence ofCGUUUAGGCAUGUUUAACU (SEQ ID NO: 430), wherein all or substantially allof the nucleotides are modified nucleotides.

In some embodiments, the sense strand of an AAT RNAi agent comprises orconsists of the nucleobase sequence of CGUUUAGGCAUGUUUAACA (SEQ ID NO:429), wherein one or more nucleotides is a modified nucleotide, and theantisense strand of an AAT RNAi agent comprises or consists of thenucleobase sequence of UGUUAAACAUGCCUAAACG (SEQ ID NO: 80), wherein oneor more nucleotides is a modified nucleotide.

In some embodiments, the sense strand of an AAT RNAi agent comprises orconsists of the nucleobase sequence of CGUUUAGGCAUGUUUAACU (SEQ ID NO:430), wherein one or more nucleotides is a modified nucleotide, and theantisense strand of an AAT RNAi agent comprises or consists of thenucleobase sequence of AGUUAAACAUGCCUAAACG (SEQ ID NO: 81), wherein oneor more nucleotides is a modified nucleotide.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGUU (SEQ IDNO: 794), wherein at least one or more nucleotides is a modifiednucleotide, and the sense strand of an AAT RNAi agent comprises orconsists of the nucleobase sequence of CGUUUAGGCAUGUUUAACAUU (SEQ ID NO:857).

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCUU (SEQ IDNO: 839), wherein at least one or more nucleotides is a modifiednucleotide, and the sense strand of an AAT RNAi agent comprises orconsists of the nucleobase sequence of GCGUUUAGGCAUGUUUAACAUU (SEQ IDNO: 885).

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCG (SEQ IDNO: 800), wherein at least one or more nucleotides is a modifiednucleotide, and the sense strand of an AAT RNAi agent comprises orconsists of the nucleobase sequence of CGCGUUUAGGCAUGUUUAACA (SEQ ID NO:864).

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCU (SEQ IDNO: 801), wherein at least one or more nucleotides is a modifiednucleotide, and the sense strand of an AAT RNAi agent comprises orconsists of the nucleobase sequence of AGCGUUUAGGCAUGUUUAACA (SEQ ID NO:866).

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGUU (SEQ IDNO: 794) differing by 0, 1, 2, or 3 nucleotides, wherein at least one ormore nucleotides is a modified nucleotide, and the sense strand of anAAT RNAi agent comprises or consists of the nucleobase sequence ofCGUUUAGGCAUGUUUAACAUU (SEQ ID NO: 857) differing by 0, 1, 2, or 3nucleotides.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCUU (SEQ IDNO: 839) differing by 0, 1, 2, or 3 nucleotides, wherein at least one ormore nucleotides is a modified nucleotide, and the sense strand of anAAT RNAi agent comprises or consists of the nucleobase sequence ofGCGUUUAGGCAUGUUUAACAUU (SEQ ID NO: 885) differing by 0, 1, 2, or 3nucleotides.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCG (SEQ IDNO: 800) differing by 0, 1, 2, or 3 nucleotides, wherein at least one ormore nucleotides is a modified nucleotide, and the sense strand of anAAT RNAi agent comprises or consists of the nucleobase sequence ofCGCGUUUAGGCAUGUUUAACA (SEQ ID NO: 864) differing by 0, 1, 2, or 3nucleotides.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCU (SEQ IDNO: 801) differing by 0, 1, 2, or 3 nucleotides, wherein at least one ormore nucleotides is a modified nucleotide, and the sense strand of anAAT RNAi agent comprises or consists of the nucleobase sequence ofAGCGUUUAGGCAUGUUUAACA (SEQ ID NO: 866) differing by 0, 1, 2, or 3nucleotides.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex structure of AD04824.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex structure of AD04825.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex structure of AD04826.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex structure of AD04827.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex structure of AD04828.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex structure of AD04829.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex structure of AD04830.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex structure of AD04831.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex AD04832.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex structure of AD04833.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex structure of AD04834.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex structure of AD04835.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex structure of AD04836.

In some embodiments, the AAT RNAi agent comprises, consists of, orconsists essentially of the duplex structure of AD04837.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACG (SEQ IDNO: 80), wherein one or more nucleotides is a modified nucleotide, andwherein SEQ ID NO: 80 is located at positions 1 to 19 (5′→3′) of theantisense strand. In some embodiments, the antisense strand of an AATRNAi agent comprises or consists of the nucleobase sequence ofUGUUAAACAUGCCUAAACG (SEQ ID NO: 80), wherein all or substantially all ofthe nucleotides are modified nucleotides, and wherein SEQ ID NO: 80 islocated at positions 1 to 19 (5′→3′) of the antisense strand.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of AGUUAAACAUGCCUAAACG (SEQ IDNO: 81), wherein one or more nucleotides is a modified nucleotide, andwherein SEQ ID NO: 81 is located at positions 1 to 19 (5′→3′) of theantisense strand. In some embodiments, the antisense strand of an AATRNAi agent comprises or consists of the nucleobase sequence ofAGUUAAACAUGCCUAAACG (SEQ ID NO: 81), wherein all or substantially all ofthe nucleotides are modified nucleotides, and wherein SEQ ID NO: 81 islocated at positions 1 to 19 (5′→3′) of the antisense strand.

In some embodiments, the sense strand of an AAT RNAi agent comprises thenucleobase sequence of CGUUUAGGCAUGUUUAACA (SEQ ID NO: 429), wherein oneor more nucleotides is a modified nucleotide, and wherein position 19 ofSEQ ID NO: 429 forms a base pair with the nucleotide located at the 5′terminal end of the antisense strand. In some embodiments, the sensestrand of an AAT RNAi agent comprises or consists of the nucleobasesequence of CGUUUAGGCAUGUUUAACA (SEQ ID NO: 429), wherein all orsubstantially all of the nucleotides are modified nucleotides, andwherein position 19 of SEQ ID NO: 429 forms a base pair with thenucleotide located at the 5′ terminal end of the antisense strand.

In some embodiments, the sense strand of an AAT RNAi agent comprises thenucleobase sequence of CGUUUAGGCAUGUUUAACU (SEQ ID NO: 430), wherein oneor more nucleotides is a modified nucleotide, and wherein position 19 ofSEQ ID NO: 430 forms a base pair with the nucleotide located at the 5′terminal end of the antisense strand. In some embodiments, the sensestrand of AN AAT RNAI agent comprises the nucleobase sequence ofCGUUUAGGCAUGUUUAACU (SEQ ID NO: 430), wherein all or substantially allof the nucleotides are modified nucleotides, and wherein position 19 ofSEQ ID NO: 430 forms a base pair with the nucleotide located at the 5′terminal end of the antisense strand.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGUU (SEQ IDNO: 794), wherein at least one or more nucleotides is a modifiednucleotide, and wherein SEQ ID NO: 794 is located at positions 1 to 21(5′→3′) of the antisense strand. In some embodiments, the antisensestrand of an AAT RNAi agent comprises or consists of the nucleobasesequence of UGUUAAACAUGCCUAAACGUU (SEQ ID NO: 794), wherein all orsubstantially all of the nucleotides are modified nucleotides, andwherein SEQ ID NO: 794 is located at positions 1 to 21 (5′→3′) of theantisense strand.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCUU (SEQ IDNO: 839), wherein at least one or more nucleotides is a modifiednucleotide, and wherein SEQ ID NO: 839 is located at positions 1 to 22(5′→3′) of the antisense strand. In some embodiments, the antisensestrand of an AAT RNAi agent comprises or consists of the nucleobasesequence of UGUUAAACAUGCCUAAACGCUU (SEQ ID NO: 839), wherein all orsubstantially all of the nucleotides are modified nucleotides, andwherein SEQ ID NO: 839 is located at positions 1 to 22 (5′→3′) of theantisense strand.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCG (SEQ IDNO: 800), wherein at least one or more nucleotides is a modifiednucleotide, and wherein SEQ ID NO: 800 is located at positions 1 to 21(5′→3′) of the antisense strand. In some embodiments, the antisensestrand of an AAT RNAi agent comprises or consists of the nucleobasesequence of UGUUAAACAUGCCUAAACGCG (SEQ ID NO: 800), wherein all orsubstantially all of the nucleotides are modified nucleotides, andwherein SEQ ID NO: 800 is located at positions 1 to 21 (5′→3′) of theantisense strand.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCU (SEQ IDNO: 801), wherein at least one or more nucleotides is a modifiednucleotide, and wherein SEQ ID NO: 801 is located at positions 1 to 21(5′→3′) of the antisense strand. In some embodiments, the antisensestrand of an AAT RNAi agent comprises or consists of the nucleobasesequence of UGUUAAACAUGCCUAAACGCU (SEQ ID NO: 801), wherein all orsubstantially all of the nucleotides are modified nucleotides, andwherein SEQ ID NO: 801 is located at positions 1 to 21 (5′→3′) of theantisense strand.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGUU (SEQ IDNO: 794), wherein at least one or more nucleotides is a modifiednucleotide, and wherein SEQ ID NO: 794 is located at the 5′ the terminalend of the antisense strand, and wherein sense strand of an AAT RNAiagent comprises or consists of the nucleobase sequence ofCGUUUAGGCAUGUUUAACAUU (SEQ ID NO: 857).

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCUU (SEQ IDNO: 839), wherein at least one or more nucleotides is a modifiednucleotide, and wherein SEQ ID NO: 839 is located at the 5′ the terminalend of the antisense strand, and the sense strand of an AAT RNAi agentcomprises or consists of the nucleobase sequence ofGCGUUUAGGCAUGUUUAACAUU (SEQ ID NO: 885).

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCG (SEQ IDNO: 800), wherein at least one or more nucleotides is a modifiednucleotide, and wherein SEQ ID NO: 800 is located at the 5′ the terminalend of the antisense strand, and the sense strand of an AAT RNAi agentcomprises or consists of the nucleobase sequence ofCGCGUUUAGGCAUGUUUAACA (SEQ ID NO: 864).

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCU (SEQ IDNO: 801), wherein at least one or more nucleotides is a modifiednucleotide, and wherein SEQ ID NO: 801 is located at the 5′ the terminalend of the antisense strand, and the sense strand of an AAT RNAi agentcomprises or consists of the nucleobase sequence ofAGCGUUUAGGCAUGUUUAACA (SEQ ID NO: 866).

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGUU (SEQ IDNO: 794) differing by 0, 1, 2, or 3 nucleotides, wherein at least one ormore nucleotides is a modified nucleotide, and wherein SEQ ID NO: 794 islocated at the 5′ the terminal end of the antisense strand, and thesense strand of an AAT RNAi agent comprises or consists of thenucleobase sequence of CGUUUAGGCAUGUUUAACAUU (SEQ ID NO: 857) differingby 0, 1, 2, or 3 nucleotides.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCUU (SEQ IDNO: 839) differing by 0, 1, 2, or 3 nucleotides, wherein at least one ormore nucleotides is a modified nucleotide, and wherein SEQ ID NO: 839 islocated at the 5′ the terminal end of the antisense strand, and thesense strand of an AAT RNAi agent comprises or consists of thenucleobase sequence of GCGUUUAGGCAUGUUUAACAUU (SEQ ID NO: 885) differingby 0, 1, 2, or 3 nucleotides.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCG (SEQ IDNO: 800) differing by 0, 1, 2, or 3 nucleotides, wherein at least one ormore nucleotides is a modified nucleotide, and wherein SEQ ID NO: 800 islocated at the 5′ the terminal end of the antisense strand, and thesense strand of an AAT RNAi agent comprises or consists of thenucleobase sequence of CGCGUUUAGGCAUGUUUAACA (SEQ ID NO: 864) differingby 0, 1, 2, or 3 nucleotides.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the nucleobase sequence of UGUUAAACAUGCCUAAACGCU (SEQ IDNO: 801) differing by 0, 1, 2, or 3 nucleotides, wherein at least one ormore nucleotides is a modified nucleotide, and wherein SEQ ID NO: 801 islocated at the 5′ the terminal end of the antisense strand, and thesense strand of an AAT RNAi agent comprises or consists of thenucleobase sequence of AGCGUUUAGGCAUGUUUAACA (SEQ ID NO: 866) differingby 0, 1, 2, or 3 nucleotides.

The AAT RNAi agents described herein can include one or more modifiednucleotides. The AAT RNAi agents described herein can also include oneor more phosphorothioate internucleoside linkages.

The AAT RNAi agents described herein can also include one or moretargeting groups or linking groups. In some embodiments, the AAT RNAiagents disclosed herein include one or more targeting groups. In someembodiments, the targeting groups are comprised of an asialoglycoproteinreceptor ligand. In some embodiments, the asialoglycoprotein receptorligand comprises a galactose or galactose-derivative cluster. In someembodiments, the galactose-derivative cluster comprisesN-acetyl-galactosamine. In some embodiments, the targeting ligandcomprises an N-acetyl-galactosamine trimer. In some embodiments, atargeting group is conjugated to the sense strand of the AAT RNAi agentsdisclosed herein.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the sequence (5′→3′) usGfsusUfaAfaCfaUfgCfcUfaAfaCfgusu(SEQ ID NO: 913), wherein a, c, g, and u are 2′-O-methyl adenosine,cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Uf are2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands is a phosphorothioate linkage, and the sense strand is at leastsubstantially complementary to the antisense strand.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the sequence (5′→3′) usGfsusUfaAfaCfaUfgCfcUfaAfaCfgcusu(SEQ ID NO: 958), wherein a, c, g, and u are 2′-O-methyl adenosine,cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Uf are2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands is a phosphorothioate linkage, and the sense strand is at leastsubstantially complementary to the antisense strand.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the sequence (5′→3′) usGfsuUfaAfaCfaUfgCfcUfaAfaCfgsCfsg(SEQ ID NO: 959), wherein a, c, g, and u are 2′-O-methyl adenosine,cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Uf are2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands is a phosphorothioate linkage, and the sense strand is at leastsubstantially complementary to the antisense strand.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the sequence (5′→3′) usGfsuUfaAfacaugCfcUfaAfaCfgCfsu(SEQ ID NO: 960), wherein a, c, g, and u are 2′-O-methyl adenosine,cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Uf are2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands is a phosphorothioate linkage, and the sense strand is at leastsubstantially complementary to the antisense strand.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the sequence (5′→3′) usGfsusUfaAfaCfaUfgCfcUfaAfaCfgusu(SEQ ID NO: 913) and the sense strand of an AAT RNAi agent comprises orconsists of the sequence (5′→3′) cguuuaGfGfCfauguuuaacausu (SEQ ID NO:1276), wherein a, c, g, and u are 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf are 2′-fluoroadenosine, cytidine, guanosine, or uridine, respectively; s is aphosphorothioate linkage; wherein optionally present on the sense strandis one, two, or more inverted abasic deoxyribose (invAb); and whereinoptionally linked to the 5′ terminal end of the sense strand is atargeting ligand that includes N-acetyl-galactosamine.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the sequence (5′→3′) usGfsusUfaAfaCfaUfgCfcUfaAfaCfgcusu(SEQ ID NO: 958) and the sense strand of an AAT RNAi agent comprises orconsists of the sequence (5′→3′) gcguuuaGfGfCfauguuuaacausu (SEQ ID NO:1277), wherein a, c, g, and u are 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf are 2′-fluoroadenosine, cytidine, guanosine, or uridine, respectively; s is aphosphorothioate linkage; wherein optionally present on the sense strandis one, two, or more inverted abasic deoxyribose (invAb); and whereinoptionally linked to the 5′ terminal end of the sense strand is atargeting ligand that includes N-acetyl-galactosamine.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the sequence (5′→3′) usGfsuUfaAfaCfaUfgCfcUfaAfaCfgsCfsg(SEQ ID NO: 959) and the sense strand of an AAT RNAi agent comprises orconsists of the sequence (5′→3′) cgcguuuaGfGfCfauguuuaaca (SEQ ID NO:1278), wherein a, c, g, and u are 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf are 2′-fluoroadenosine, cytidine, guanosine, or uridine, respectively; s is aphosphorothioate linkage; wherein optionally present on the sense strandis one, two, or more inverted abasic deoxyribose (invAb); and whereinoptionally linked to the 5′ terminal end of the sense strand is atargeting ligand that includes N-acetyl-galactosamine.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the sequence (5′→3′) usGfsuUfaAfacaugCfcUfaAfaCfgCfsu(SEQ ID NO: 960) and the sense strand of an AAT RNAi agent comprises orconsists of the sequence (5′→3′) agcguuuaGfGfCfauguuuaaca (SEQ ID NO:1279), wherein a, c, g, and u are 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf are 2′-fluoroadenosine, cytidine, guanosine, or uridine, respectively; s is aphosphorothioate linkage; wherein optionally present on the sense strandis one, two, or more inverted abasic deoxyribose (invAb); and whereinoptionally linked to the 5′ terminal end of the sense strand is atargeting ligand that includes N-acetyl-galactosamine.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the sequence (5′→3′) usGfsusUfaAfaCfaUfgCfcUfaAfaCfgusu(SEQ ID NO: 913) and the sense strand of an AAT RNAi agent comprises orconsists of (5′→3′) (NAG37)s(invAb)scguuuaGfGfCfauguuuaacausu(invAb)(SEQ ID NO: 1028), wherein a, c, g, and u are 2′-O-methyl adenosine,cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Uf are2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; s isa phosphorothioate linkage; (invAb) is inverted abasic deoxyribose(invAb); and (NAG37) is the targeting ligand that includesN-acetyl-galactosamine having the structure shown in Table 7 herein.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the sequence (5′→3′) usGfsusUfaAfaCfaUfgCfcUfaAfaCfgcusu(SEQ ID NO: 958) and the sense strand of an AAT RNAi agent comprises orconsists of the sequence (5′→3′)(NAG37)s(invAb)sgcguuuaGfGfCfauguuuaacausu(invAb) (SEQ ID NO: 1030),wherein a, c, g, and u are 2′-O-methyl adenosine, cytidine, guanosine,or uridine, respectively; Af, Cf, Gf, and Uf are 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; s is a phosphorothioatelinkage; (invAb) is inverted abasic deoxyribose (invAb); and (NAG37) isthe targeting ligand that includes N-acetyl-galactosamine having thestructure shown in Table 7 herein.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the sequence (5′→3′) usGfsuUfaAfaCfaUfgCfcUfaAfaCfgsCfsg(SEQ ID NO: 959) and the sense strand of an AAT RNAi agent comprises orconsists of the sequence (5′→3′)(NAG37)s(invAb)scgcguuuaGfGfCfauguuuaacas(invAb) (SEQ ID NO: 1024),wherein a, c, g, and u are 2′-O-methyl adenosine, cytidine, guanosine,or uridine, respectively; Af, Cf, Gf, and Uf are 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; s is a phosphorothioatelinkage; (invAb) is inverted abasic deoxyribose (invAb); and (NAG37) isthe targeting ligand that includes N-acetyl-galactosamine having thestructure shown in Table 7 herein.

In some embodiments, the antisense strand of an AAT RNAi agent comprisesor consists of the sequence (5′→3′) usGfsuUfaAfacaugCfcUfaAfaCfgCfsu(SEQ ID NO: 960) and the sense strand of an AAT RNAi agent comprises orconsists of the sequence (5′→3′)(NAG37)s(invAb)sagcguuuaGfGfCfauguuuaacas(invAb) (SEQ ID NO: 1033),wherein a, c, g, and u are 2′-O-methyl adenosine, cytidine, guanosine,or uridine, respectively; Af, Cf, Gf, and Uf are 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; s is a phosphorothioatelinkage; (invAb) is inverted abasic deoxyribose (invAb); and (NAG37) isthe targeting ligand that includes N-acetyl-galactosamine having thestructure shown in Table 7 herein.

In some embodiments, the AAT RNAi agents described herein can includeone or more targeting groups having the structure of (PAZ), (NAG25),(NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s,(NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32),(NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s,(NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39),(NAG39)s, as defined herein in Table 7.

In some embodiments, the AAT RNAi agents described herein include onetargeting group at the 5′ end of the sense strand having the structureof (PAZ), (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s,(NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31),(NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s,(NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38),(NAG38)s, (NAG39), (NAG39)s, as defined herein in Table 7.

The AAT RNAi agents disclosed herein can be incorporated into acomposition comprising one or more disclosed AAT RNAi agent and at leastone pharmaceutically acceptable excipient.

In some embodiments, the compositions disclosed herein comprising one ormore of the disclosed AAT RNAi agents and at least one pharmaceuticallyacceptable excipient is a pharmaceutical composition.

The pharmaceutical compositions comprising one or more AAT RNAi agentscan be administered in a number of ways depending upon whether local orsystemic treatment is desired. Administration can be, but is not limitedto, intravenous, intraarterial, subcutaneous, intraperitoneal, subdermal(e.g., via an implanted device), and intraparenchymal administration. Insome embodiments, the pharmaceutical compositions described herein areadministered by subcutaneous injection.

In some embodiments, the compositions comprising one or more disclosedAAT RNAi agents and at least one pharmaceutically acceptable excipientcan further comprise one or more additional therapeutics or treatments.

In some embodiments, the compositions described herein comprising one ormore AAT RNAi agents are packaged in a kit, container, pack, dispenser,pre-filled syringes, or vials. In some embodiments, the compositionsdescribed herein are administered parenterally.

The AAT RNAi agents and compositions comprising same that are disclosedherein can be administered to a subject to inhibit the expression of thealpha-1 antitrypsin gene in the subject. In some embodiments, thesubject is a human. In some embodiments, the subject is a human that hasbeen diagnosed with having AATD.

In some embodiments, disclosed herein are methods for inhibitingexpression of an AAT gene in a cell, the methods comprisingadministering an AAT RNAi agent that has an antisense strand that is atleast partially complementary to the portion of an AAT mRNA having anyone of the sequences listed in Table 1.

The AAT RNAi agents and compositions comprising same disclosed hereinmay be administered to a subject for the treatment of AATD (including acondition or disease caused by alpha-1 antitrypsin deficiency). Thecondition or disease that may be treated, prevented, and/or managed byadministration of the AAT RNAi agents and compositions comprising samedisclosed herein include chronic hepatitis, cirrhosis, hepatocellularcarcinoma, transaminitis, cholestasis, fibrosis, or fulminant hepaticfailure.

As used herein, the terms “oligonucleotide” and “polynucleotide” mean apolymer of linked nucleosides each of which can be independentlymodified or unmodified.

As used herein, an “RNAi agent” or “RNAi trigger” means a compositionthat contains an RNA or RNA-like (e.g., chemically modified RNA)oligonucleotide molecule that is capable of degrading or inhibitingtranslation of messenger RNA (mRNA) transcripts of a target mRNA in asequence specific manner. As used herein, RNAi agents may operatethrough the RNA interference mechanism (i.e., inducing RNA interferencethrough interaction with the RNA interference pathway machinery(RNA-induced silencing complex or RISC) of mammalian cells), or by anyalternative mechanism(s) or pathway(s). While it is believed that RNAiagents, as that term is used herein, operate primarily through the RNAinterference mechanism, the disclosed RNAi agents are not bound by orlimited to any particular pathway or mechanism of action. RNAi agentsdisclosed herein are comprised of a sense strand and an antisensestrand, and include, but are not limited to: short interfering RNAs(siRNAs), double-strand RNAs (dsRNA), micro RNAs (miRNAs), short hairpinRNAs (shRNA), and dicer substrates. The antisense strand of the RNAiagents described herein is at least partially complementary to the mRNAbeing targeted (e.g. AAT mRNA). RNAi agents can include one or moremodified nucleotides and/or one or more non-phosphodiester linkages.

As used herein, the terms “silence,” “reduce,” “inhibit,”“down-regulate,” or “knockdown” when referring to expression of a givengene, mean that the expression of the gene, as measured by the level ofRNA transcribed from the gene or the level of polypeptide, protein orprotein subunit translated from the mRNA in a cell, group of cells,tissue, organ, or subject in which the gene is transcribed, is reducedwhen the cell, group of cells, tissue, organ, or subject is treated withthe RNAi agents described herein as compared to a second cell, group ofcells, tissue, organ, or subject that has not or have not been sotreated.

As used herein, the terms “sequence” and “nucleotide sequence” mean asuccession or order of nucleobases or nucleotides, described with asuccession of letters using standard nomenclature.

As used herein, a “base”, “nucleotide base,” or “nucleobase,” is aheterocyclic pyrimidine or purine compound, which is a standardconstituent of all nucleic acids, and includes the bases that form thenucleotides adenine (A), guanine (G), cytosine (C), thymine (T), anduracil (U). A nucleobase may further be modified to include, withoutlimitation, universal bases, hydrophobic bases, promiscuous bases,size-expanded bases, and fluorinated bases. As used herein, the term“nucleotide” can include a modified nucleotide (such as, for example, anucleotide mimic, abasic residue (Ab), or a surrogate replacementmoiety).

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleobase or nucleotidesequence (e.g., RNAi agent sense strand or targeted mRNA) in relation toa second nucleobase or nucleotide sequence (e.g., RNAi agent antisensestrand or a single-stranded antisense oligonucleotide), means theability of an oligonucleotide or polynucleotide including the firstnucleotide sequence to hybridize (form base pair hydrogen bonds undermammalian physiological conditions (or similar conditions in vitro)) andform a duplex or double helical structure under certain standardconditions with an oligonucleotide or polynucleotide including thesecond nucleotide sequence. Complementary sequences include Watson-Crickbase pairs or non-Watson-Crick base pairs and include natural ormodified nucleotides or nucleotide mimics, at least to the extent thatthe above hybridization requirements are fulfilled. Sequence identity orcomplementarity is independent of modification. For example, a and Af,as defined herein, are complementary to U (or T) and identical to A forthe purposes of determining identity or complementarity.

As used herein, “perfectly complementary” or “fully complementary” meansthat all (100%) of the nucleobases or nucleotides in a contiguoussequence of a first polynucleotide will hybridize with the same numberof nucleobases or nucleotides in a contiguous sequence of a secondpolynucleotide. The contiguous sequence may comprise all or a part of afirst or second nucleotide sequence.

As used herein, “partially complementary” means that in a hybridizedpair of nucleobase sequences, at least 70%, but not all, of the bases ina contiguous sequence of a first polynucleotide will hybridize with thesame number of bases in a contiguous sequence of a secondpolynucleotide.

As used herein, “substantially complementary” means that in a hybridizedpair of nucleobase sequences, at least 85%, but not all, of the bases ina contiguous sequence of a first polynucleotide will hybridize with thesame number of bases in a contiguous sequence of a secondpolynucleotide. The terms “complementary,” “fully complementary,”“partially complementary,” and “substantially complementary” herein areused with respect to the nucleobase or nucleotide matching between thesense strand and the antisense strand of an RNAi agent, or between theantisense strand of an RNAi agent and a sequence of an AAT mRNA.

As used herein, the term “substantially identical” or “substantiallyidentity” as applied to nucleic acid sequence means that a nucleic acidsequence comprises a sequence that has at least about 85% sequenceidentity or more, e.g., at least 90%, at least 95%, or at least 99%identity, compared to a reference sequence. Percentage of sequenceidentity is determined by comparing two optimally aligned sequences overa comparison window. The percentage is calculated by determining thenumber of positions at which the identical nucleic acid base occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. The inventions disclosed hereinencompass nucleotide sequences substantially identical to thosedisclosed herein.

As used herein, the terms “treat,” “treatment,” and the like, mean themethods or steps taken to provide relief from or alleviation of thenumber, severity, and/or frequency of one or more symptoms of a diseasein a subject. As used herein, “treat” and treatment” may include theprevention, management, prophylactic treatment, and/or inhibition of thenumber, severity, and/or frequency of one or more symptoms of a diseasein a subject.

As used herein, the phrase “introducing into a cell,” when referring toan RNAi agent, means functionally delivering the RNAi agent into a cell.The phrase “functional delivery,” means that delivering the RNAi agentto the cell in a manner that enables the RNAi agent to have the expectedbiological activity, e.g., sequence-specific inhibition of geneexpression.

Unless stated otherwise, use of the symbol

as used herein means that any group or groups may be linked thereto thatis in accordance with the scope of the inventions described herein.

As used herein, the term “isomers” refers to compounds that haveidentical molecular formulae, but that differ in the nature or thesequence of bonding of their atoms or in the arrangement of their atomsin space. Isomers that differ in the arrangement of their atoms in spaceare termed “stereoisomers.” Stereoisomers that are not mirror images ofone another are termed “diastereoisomers,” and stereoisomers that arenon-superimposable mirror images are termed “enantiomers,” or sometimesoptical isomers. A carbon atom bonded to four non-identical substituentsis termed a “chiral center.”

As used herein, unless specifically identified in a structure as havinga particular conformation, for each structure in which asymmetriccenters are present and thus give rise to enantiomers, diastereomers, orother stereoisomeric configurations, each structure disclosed herein isintended to represent all such possible isomers, including theiroptically pure and racemic forms. For example, the structures disclosedherein are intended to cover mixtures of diastereomers as well as singlestereoisomers.

As used in a claim herein, the phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. When used in aclaim herein, the phrase “consisting essentially of” limits the scope ofa claim to the specified materials or steps and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention.

The person of ordinary skill in the art would readily understand andappreciate that the compounds and compositions disclosed herein may havecertain atoms (e.g., N, O, or S atoms) in a protonated or deprotonatedstate, depending upon the environment in which the compound orcomposition is placed. Accordingly, as used herein, the structuresdisclosed herein envisage that certain functional groups, such as, forexample, OH, SH, or NH, may be protonated or deprotonated. Thedisclosure herein is intended to cover the disclosed compounds andcompositions regardless of their state of protonation based on theenvironment (such as pH), as would be readily understood by the personof ordinary skill in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

In addition, the materials, methods, and examples are illustrative onlyand not intended to be limiting.

Other objects, features, aspects, and advantages of the invention willbe apparent from the following detailed description, accompanyingfigures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1E represent the chemical duplex structure of AD04828 shownas a sodium salt.

FIGS. 2A to 2E represent the chemical duplex structure of AD04828 shownas a free acid.

FIGS. 3A to 3E represent the chemical duplex structure of AD04831 shownas a sodium salt.

FIG. 4A to 4E represent the chemical duplex structure of AD04831 shownas a free acid.

FIG. 5A to 5E represent the chemical duplex structure of AD04836 shownas a sodium salt.

FIG. 6A to 6E represent the chemical duplex structure of AD04836 shownas a free acid.

FIG. 7A to 7E represent the chemical duplex structure of AD04837 shownas a sodium salt.

FIG. 8A to 8E represent the chemical duplex structure of AD04837 shownas a free acid.

FIG. 9 is a bar graph showing average normalized cynomolgus monkey(cyno) AAT (cAAT) serum levels in cynos (n=3) following a singlesubcutaneous administration of 3 mg/kg of either AD04824, AD04825,AD04826, or AD04827, according to Example 4. AAT serum levels werenormalized to average pre-treatment values. Experimental error is shownas standard deviation.

FIG. 10 is a bar graph showing average normalized cAAT serum levels incynos (n=2 or n=3) following a single subcutaneous administration of 3mg/kg of either AD04828, AD04836, AD04831, or AD04837, according toExample 5. AAT serum levels were normalized to average pre-treatmentvalues. Experimental error is shown as standard deviation.

FIG. 11. is a bar graph showing the results of a western blot analysisof the soluble fractions (Z-AAT monomer) from livers of PiZ mice dosedwith either saline or NAG-conjugated AAT RNAi agent having the duplexstructure AD04837, dosed for 8 weeks q2w, normalized to baselinecontrol, according to Example 7. Individual mouse measurements are showngrouped by treatment group, and experimental error is shown as standarddeviation.

FIG. 12. is a bar graph showing the results of a western blot analysisof the insoluble fractions (Z-AAT polymer) from livers of PiZ mice dosedwith either saline or NAG-conjugated AAT RNAi agent having the duplexstructure AD04837, according to Example 7. Individual mouse measurementsare shown grouped by treatment group, and experimental error is shown asstandard deviation.

DETAILED DESCRIPTION

RNAi Agents

Described herein are RNAi agents for inhibiting expression of an AATgene (referred to herein as AAT RNAi agents or AAT RNAi triggers). EachAAT RNAi agent comprises a sense strand and an antisense strand. Thesense strand and the antisense strand each can be 16 to 30 nucleotidesin length. In some embodiments, the sense and antisense strands each canbe 17 to 26 nucleotides in length. The sense and antisense strands canbe either the same length or they can be different lengths. In someembodiments, the sense and antisense strands are each independently17-21 nucleotides in length. In some embodiments, the sense andantisense strands are each 21-26 nucleotides in length. In someembodiments, the sense and antisense strands are each 21-24 nucleotidesin length. In some embodiments, the sense strand is about 19 nucleotidesin length while the antisense strand is about 21 nucleotides in length.In some embodiments, the sense strand is about 21 nucleotides in lengthwhile the antisense strand is about 23 nucleotides in length. In someembodiments, a sense strand is 23 nucleotides in length and an antisensestrand is 21 nucleotides in length. In some embodiments, both the senseand antisense strands are each 21 nucleotides in length. In someembodiments, a sense strand is 22 nucleotides in length and an antisensestrand is 21 nucleotides in length. In some embodiments, a sense strandis 19 nucleotides in length and an antisense strand is 21 nucleotides inlength. In some embodiments, the RNAi agent sense and antisense strandsare each independently 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26nucleotides in length. In some embodiments, a double-stranded RNAi agenthas a duplex length of about 16, 17, 18, 19, 20, 21, 22, 23 or 24nucleotides.

In some embodiments, the region of perfect or substantialcomplementarity between the sense strand and the antisense strand is16-26 (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotidesin length and occurs at or near the 5′ end of the antisense strand(e.g., this region may be separated from the 5′ end of the antisensestrand by 0, 1, 2, 3, or 4 nucleotides that are not perfectly orsubstantially complementary).

The sense strand and antisense strand each contain a core stretchsequence that is 16 to 23 nucleobases in length. An antisense strandcore stretch sequence is 100% (perfectly) complementary or at leastabout 85% (substantially) complementary to a nucleotide sequence(sometimes referred to, e.g., as a target sequence) present in the AATmRNA target. A sense strand core stretch sequence is 100% (perfectly)complementary or at least about 85% (substantially) complementary to acore stretch sequence in the antisense strand, and thus the sense strandcore stretch sequence is perfectly identical or at least about 85%identical to a nucleotide sequence (target sequence) present in the AATmRNA target. A sense strand core stretch sequence can be the same lengthas a corresponding antisense core sequence or it can be a differentlength. In some embodiments, the antisense strand core stretch sequenceis 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In someembodiments, the sense strand core stretch sequence is 16, 17, 18, 19,20, 21, 22, or 23 nucleotides in length.

Examples of nucleotide sequences used in forming AAT RNAi agents areprovided in Tables 2, 3, 4 and 5. Examples of AAT RNAi agent duplexes,that include the sense strand and antisense strand sequences in Tables2, 3, 4, and 5, are shown in Table 6.

The AAT RNAi agent sense and antisense strands anneal to form a duplex.A sense strand and an antisense strand of an AAT RNAi agent may bepartially, substantially, or fully complementary to each other. Withinthe complementary duplex region, the sense strand core stretch sequenceis at least 85% complementary or 100% complementary to the antisensecore stretch sequence. In some embodiments, the sense strand corestretch sequence contains a sequence of at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, or atleast 23 nucleotides that is at least 85% or 100% complementary to acorresponding 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide sequence ofthe antisense strand core stretch sequence (i.e., the sense andantisense core stretch sequences of an AAT RNAi agent have a region ofat least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, or at least 23 nucleotides that is at least 85%base paired or 100% base paired.)

In some embodiments, the antisense strand of an AAT RNAi agent disclosedherein differs by 0, 1, 2, or 3 nucleotides from any of the antisensestrand sequences in Table 2, Table 3, or Table 4. In some embodiments,the sense strand of an AAT RNAi agent disclosed herein differs by 0, 1,2, or 3 nucleotides from any of the sense strand sequences in Table 2,Table 3, or Table 5.

The sense strand and/or the antisense strand may optionally andindependently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides(extension) at the 3′ end, the 5′ end, or both the 3′ and 5′ ends of thecore stretch sequences. The antisense strand additional nucleotides, ifpresent, may or may not be complementary to the corresponding sequencein an AAT mRNA. The sense strand additional nucleotides, if present, mayor may not be identical to the corresponding sequence in an AAT mRNA.The antisense strand additional nucleotides, if present, may or may notbe complementary to the corresponding sense strand's additionalnucleotides, if present.

As used herein, an extension comprises 1, 2, 3, 4, 5, or 6 nucleotidesat the 5′ and/or 3′ end of the sense strand core stretch sequence and/orantisense strand core stretch sequence. The extension nucleotides on asense strand may or may not be complementary to nucleotides, either corestretch sequence nucleotides or extension nucleotides, in thecorresponding antisense strand. Conversely, the extension nucleotides onan antisense strand may or may not be complementary to nucleotides,either core stretch nucleotides or extension nucleotides, in thecorresponding sense strand. In some embodiments, both the sense strandand the antisense strand of an RNAi agent contain 3′ and 5′ extensions.In some embodiments, one or more of the 3′ extension nucleotides of onestrand base pairs with one or more 5′ extension nucleotides of the otherstrand. In other embodiments, one or more of 3′ extension nucleotides ofone strand do not base pair with one or more 5′ extension nucleotides ofthe other strand. In some embodiments, an AAT RNAi agent has anantisense strand having a 3′ extension and a sense strand having a 5′extension.

In some embodiments, an AAT RNAi agent comprises an antisense strandhaving a 3′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. Inother embodiments, an AAT RNAi agent comprises an antisense strandhaving a 3′ extension of 1, 2, or 3 nucleotides in length. In someembodiments, one or more of the antisense strand extension nucleotidescomprise uracil or thymidine nucleotides or nucleotides that arecomplementary to the corresponding AAT mRNA sequence. In someembodiments, a 3′ antisense strand extension includes or consists of oneof the following sequences, but is not limited to: AUA, UGCUU, CUG, UG,UGCC, CUGCC, CGU, CUU, UGCCUA, CUGCCU, UGCCU, UGAUU, GCCUAU, T, TT, U,UU (each listed 5′→3′).

In some embodiments, the 3′ end of the antisense strand can includeadditional abasic residues (Ab). An “abasic residue” or “abasic site” isa nucleotide or nucleoside that lacks a nucleobase at the 1′ position ofthe sugar. In some embodiments, Ab or AbAb can be added to the 3′ end ofthe antisense strand. In some embodiments, the abasic residue(s) can beadded as inverted abasic residues (invAb) (see Table 7). (See, e.g., F.Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16).

In some embodiments, an AAT RNAi agent comprises a sense strand having a3′ extension of 1, 2, 3, 4, or 5 nucleotides in length. In someembodiments, one or more of the sense strand extension nucleotidescomprises adenosine, uracil, or thymidine nucleotides, AT dinucleotide,or nucleotides that correspond to nucleotides in the AAT mRNA sequence.In some embodiments, the 3′ sense strand extension includes or consistsof one of the following sequences, but is not limited to: T, UT, TT, UU,UUT, TTT, or TTTT (each listed 5′ to 3′).

In some embodiments, the 3′ end of the sense strand may includeadditional abasic residues. In some embodiments, UUAb, UAb, or Ab areadded to the 3′ end of the sense strand. In some embodiments, the one ormore abasic residues added to the 3′ end of the sense strand areinverted (invAb). In some embodiments, one or more inverted abasicresidues or abasic sites may be inserted between the targeting ligandand the nucleobase sequence of the sense strand of the RNAi agent. Insome embodiments, the inclusion of one or more inverted abasic residuesor abasic sites at or near the terminal end or terminal ends of thesense strand of an RNAi agent allows for enhanced activity or otherdesired properties of an RNAi agent.

In some embodiments, an AAT RNAi agent comprises a sense strand having a5′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In someembodiments, one or more of the sense strand extension nucleotidescomprise uracil or adenosine nucleotides or nucleotides that correspondto nucleotides in the AAT mRNA sequence. In some embodiments, the sensestrand 5′ extension is one of the following sequences, but is notlimited to: CA, AUAGGC, AUAGG, AUAG, AUA, A, AA, AC, GCA, GGCA, GGC,UAUCA, UAUC, UCA, UAU, U, UU (each listed 5′ to 3′). A sense strand canhave a 3′ extension and/or a 5′ extension.

In some embodiments, the 5′ end of the sense strand can include one ormore additional abasic residues (e.g., (Ab) or (AbAb)). In someembodiments, the one or more abasic residues added to the 5′ end of thesense strand can be inverted (e.g., invAb). In some embodiments, one ormore inverted abasic residues can be inserted between the targetingligand and the nucleobase sequence of the sense strand of the RNAiagent. In some embodiments, the inclusion of one or more inverted abasicresidues at or near the terminal end or terminal ends of the sensestrand of an RNAi agent may allow for enhanced activity or other desiredproperties of an RNAi agent. In some embodiments, an abasic(deoxyribose) residue can be replaced with a ribitol (abasic ribose)residue.

In some embodiments, the 3′ end of the antisense strand core stretchsequence, or the 3′ end of the antisense strand sequence, may include aninverted abasic residue (invAb (see Table 7)).

Examples of sequences used in forming AAT RNAi agents are provided inTables 2, 3, 4, and 5. In some embodiments, an AAT RNAi agent antisensestrand includes a sequence of any of the sequences in Tables 2, 3, or 4.In some embodiments, an AAT RNAi agent antisense strand includes thesequence of nucleotides (from 5′ end→3′ end) 1-17, 2-15, 2-17, 1-18,2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24,or 2-24, of any of the sequences in Table 2, Table 3, or Table 4. Incertain embodiments, an AAT RNAi agent antisense strand comprises orconsists of a modified sequence of any one of the modified sequences inTable 4. In some embodiments, an AAT RNAi agent sense strand includesthe sequence of any of the sequences in Tables 2, 3, or 5. In someembodiments, an AAT RNAi agent sense strand includes the sequence ofnucleotides (from 5′ end→3′ end) 1-18, 1-19, 1-20, 1-21, 1-22, 1-23,1-24, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 3-20, 3-21, 3-22, 3-23, 3-24,4-21, 4-22, 4-23, 4-24, 5-22, 5-23, 5-24, 6-23, 6-24, 7-24, of any ofthe sequences in Tables 2, 3, or 5. In certain embodiments, an AAT RNAiagent sense strand comprises or consists of a modified sequence of anyone of the modified sequences in Table 5.

In some embodiments, the sense and antisense strands of the RNAi agentsdescribed herein contain the same number of nucleotides. In someembodiments, the sense and antisense strands of the RNAi agentsdescribed herein contain different numbers of nucleotides. In someembodiments, the sense strand 5′ end and the antisense strand 3′ end ofan RNAi agent form a blunt end. In some embodiments, the sense strand 3′end and the antisense strand 5′ end of an RNAi agent form a blunt end.In some embodiments, both ends of an RNAi agent form blunt ends. In someembodiments, neither end of an RNAi agent is blunt-ended. As used hereina blunt end refers to an end of a double stranded RNAi agent in whichthe terminal nucleotides of the two annealed strands are complementary(form a complementary base-pair).

In some embodiments, the sense strand 5′ end and the antisense strand 3′end of an RNAi agent form a frayed end. In some embodiments, the sensestrand 3′ end and the antisense strand 5′ end of an RNAi agent form afrayed end. In some embodiments, both ends of an RNAi agent form afrayed end. In some embodiments, neither end of an RNAi agent is afrayed end. As used herein a frayed end refers to an end of a doublestranded RNAi agent in which the terminal nucleotides of the twoannealed strands from a pair (i.e., do not form an overhang) but are notcomplementary (i.e. form a non-complementary pair). As used herein, anoverhang is a stretch of one or more unpaired nucleotides at the end ofone strand of a double stranded RNAi agent. The unpaired nucleotides maybe on the sense strand or the antisense strand, creating either 3′ or 5′overhangs. In some embodiments, the RNAi agent contains: a blunt end anda frayed end, a blunt end and 5′ overhang end, a blunt end and a 3′overhang end, a frayed end and a 5′ overhang end, a frayed end and a 3′overhang end, two 5′ overhang ends, two 3′ overhang ends, a 5′ overhangend and a 3′ overhang end, two frayed ends, or two blunt ends.

Modified nucleotides, when used in various polynucleotide oroligonucleotide constructs, can preserve activity of the compound incells while at the same time increasing the serum stability of thesecompounds, and can also minimize the possibility of activatinginterferon activity in humans upon administering of the polynucleotideor oligonucleotide construct.

In some embodiments, an AAT RNAi agent is prepared or provided as asalt, mixed salt, or a free-acid. In some embodiments, an AAT RNAi agentis prepared as a sodium salt. Such forms are within the scope of theinventions disclosed herein.

Modified Nucleotides

In some embodiments, an AAT RNAi agent contains one or more modifiednucleotides. As used herein, a “modified nucleotide” is a nucleotideother than a ribonucleotide (2′-hydroxyl nucleotide). In someembodiments, at least 50% (e.g., at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 97%, at least 98%, at least99%, or 100%) of the nucleotides are modified nucleotides. As usedherein, modified nucleotides include, but are not limited to,deoxyribonucleotides, nucleotide mimics, abasic nucleotides (representedherein as Ab), 2′-modified nucleotides, 3′ to 3′ linkages (inverted)nucleotides (represented herein as invdN, invN, invn), modifiednucleobase-comprising nucleotides, bridged nucleotides, peptide nucleicacids (PNAs), 2′,3′-seco nucleotide mimics (unlocked nucleobaseanalogues, represented herein as N_(UNA or) NUNA), locked nucleotides(represented herein as N_(LNA) or NLNA), 3′-O-methoxy (2′internucleoside linked) nucleotides (represented herein as 3′-OMen),2′-F-Arabino nucleotides (represented herein as NfANA or Nf_(ANA)),5′-Me, 2′-fluoro nucleotide (represented herein as 5Me-Nf), morpholinonucleotides, vinyl phosphonate deoxyribonucleotides (represented hereinas vpdN), vinyl phosphonate containing nucleotides, and cyclopropylphosphonate containing nucleotides (cPrpN). 2′-modified nucleotides(i.e. a nucleotide with a group other than a hydroxyl group at the 2′position of the five-membered sugar ring) include, but are not limitedto, 2′-O-methyl nucleotides (represented herein as a lower case letter‘n’ in a nucleotide sequence), 2′-deoxy-2′-fluoro nucleotides(represented herein as Nf, also represented herein as 2′-fluoronucleotide), 2′-deoxy nucleotides (represented herein as dN),2′-methoxyethyl (2′-O-2-methoxylethyl) nucleotides (represented hereinas NM or 2′-MOE), 2′-amino nucleotides, and 2′-alkyl nucleotides. It isnot necessary for all positions in a given compound to be uniformlymodified. Conversely, more than one modification can be incorporated ina single AAT RNAi agent or even in a single nucleotide thereof. The AATRNAi agent sense strands and antisense strands can be synthesized and/ormodified by methods known in the art. Modification at one nucleotide isindependent of modification at another nucleotide.

Modified nucleobases include synthetic and natural nucleobases, such as5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl(e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives ofadenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or2-n-butyl) and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine,5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine,6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adeninesand guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine,7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

In some embodiments, all or substantially all of the nucleotides of anRNAi agent are modified nucleotides. As used herein, an RNAi agentwherein substantially all of the nucleotides present are modifiednucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or4) nucleotides in both the sense strand and the antisense strand beingribonucleotides (i.e., unmodified). As used herein, a sense strandwherein substantially all of the nucleotides present are modifiednucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2)nucleotides in the sense strand being ribonucleotides. As used herein,an antisense sense strand wherein substantially all of the nucleotidespresent are modified nucleotides is an antisense strand having two orfewer (i.e., 0, 1, or 2) nucleotides in the sense strand beingribonucleotides. In some embodiments, one or more nucleotides of an RNAiagent is a ribonucleotide.

Modified Internucleoside Linkages

In some embodiments, one or more nucleotides of an AAT RNAi agent arelinked by non-standard linkages or backbones (i.e., modifiedinternucleoside linkages or modified backbones). Modifiedinternucleoside linkages or backbones include, but are not limited to,5′-phosphorothioate groups (represented herein as a lower case “s”),chiral phosphorothioates, thiophosphates, phosphorodithioates,phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g.,methyl phosphonates or 3′-alkylene phosphonates), chiral phosphonates,phosphinates, phosphoramidates (e.g., 3′-amino phosphoramidate,aminoalkylphosphoramidates, or thionophosphoramidates),thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholinolinkages, boranophosphates having normal 3′-5′ linkages, 2′-5′ linkedanalogs of boranophosphates, or boranophosphates having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. In some embodiments, a modifiedinternucleoside linkage or backbone lacks a phosphorus atom. Modifiedinternucleoside linkages lacking a phosphorus atom include, but are notlimited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixedheteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or moreshort chain heteroatomic or heterocyclic inter-sugar linkages. In someembodiments, modified internucleoside backbones include, but are notlimited to, siloxane backbones, sulfide backbones, sulfoxide backbones,sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones,sulfamate backbones, methyleneimino and methylenehydrazino backbones,sulfonate and sulfonamide backbones, amide backbones, and otherbackbones having mixed N, O, S, and CH₂ components.

In some embodiments, a sense strand of an AAT RNAi agent can contain 1,2, 3, 4, 5, or 6 phosphorothioate linkages, an antisense strand of anAAT RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioatelinkages, or both the sense strand and the antisense strandindependently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages.In some embodiments, a sense strand of an AAT RNAi agent can contain 1,2, 3, or 4 phosphorothioate linkages, an antisense strand of an AAT RNAiagent can contain 1, 2, 3, or 4 phosphorothioate linkages, or both thesense strand and the antisense strand independently can contain 1, 2, 3,or 4 phosphorothioate linkages.

In some embodiments, an AAT RNAi agent sense strand contains at leasttwo phosphorothioate internucleoside linkages. In some embodiments, theat least two phosphorothioate internucleoside linkages are between thenucleotides at positions 1-3 from the 3′ end of the sense strand. Insome embodiments, the at least two phosphorothioate internucleosidelinkages are between the nucleotides at positions 1-3, 2-4, 3-5, 4-6,4-5, or 6-8 from the 5′ end of the sense strand. In some embodiments, anAAT RNAi agent antisense strand contains four phosphorothioateinternucleoside linkages. In some embodiments, the four phosphorothioateinternucleoside linkages are between the nucleotides at positions 1-3from the 5′ end of the antisense strand and between the nucleotides atpositions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5′ end.In some embodiments, an AAT RNAi agent contains at least twophosphorothioate internucleoside linkages in the sense strand and threeor four phosphorothioate internucleoside linkages in the antisensestrand.

In some embodiments, an AAT RNAi agent contains one or more modifiednucleotides and one or more modified internucleoside linkages. In someembodiments, a 2′-modified nucleoside is combined with modifiedinternucleoside linkage.

AAT RNAi Agents

In some embodiments, the AAT RNAi agents disclosed herein target an AATgene at or near the positions of the AAT gene shown in Table 1. In someembodiments, the antisense strand of an AAT RNAi agent disclosed hereinincludes a core stretch sequence that is fully, substantially, or atleast partially complementary to a target AAT 19-mer sequence disclosedin Table 1.

TABLE 1 AAT 19-mer mRNA Target Sequences (taken from human AAT cDNA, GenBank NM_000295.4 (SEQ ID  NO: 1)) Corresponding SEQAAT 19-mer Gene Position ID Target Sequences (taken from  NO: (5′→3′)SEQ ID NO: 1)  2 CGUUUAGGCAUGUUUAACA 1000-1018  3 AACAGCACCAAUAUCUUCU 469-487  4 AUAUCAUCACCAAGUUCCU 1142-1160  5 AGAUGCUGCCCAGAAGACA 348-366  6 CUGGCACACCAGUCCAACA  454-472  7 UGGCACACCAGUCCAACAG  455-473 8 GCACACCAGUCCAACAGCA  457-475  9 CAGUCCAACAGCACCAAUA  463-481 10AGUCCAACAGCACCAAUAU  464-482 11 GUCCAACAGCACCAAUAUC  465-483 12CCAACAGCACCAAUAUCUU  467-485 13 CCCCAGUGAGCAUCGCUAC  491-509 14GAGCAUCGCUACAGCCUUU  498-516 15 GCAUCGCUACAGCCUUUGC  500-518 16CAUCGCUACAGCCUUUGCA  501-519 17 UCGCUACAGCCUUUGCAAU  503-521 18CUACAGCCUUUGCAAUGCU  506-524 19 ACAGCCUUUGCAAUGCUCU  508-526 20GAAGGCUUCCAGGAACUCC  613-631 21 UAGUGGAUAAGUUUUUGGA  710-728 22UGUACCACUCAGAAGCCUU  743-761 23 GUACCACUCAGAAGCCUUC  744-762 24ACACCGAAGAGGCCAAGAA  779-797 25 ACCGAAGAGGCCAAGAAAC  781-799 26AGGCCAAGAAACAGAUCAA  788-806 27 GGCCAAGAAACAGAUCAAC  789-807 28GCCAAGAAACAGAUCAACG  790-808 29 UACUCAAGGGAAAAUUGUG  825-843 30CUCAAGGGAAAAUUGUGGA  827-845 31 UCAAGGGAAAAUUGUGGAU  828-846 32UUGGUCAAGGAGCUUGACA  847-865 33 AGGAGCUUGACAGAGACAC  854-872 34AGCUUGACAGAGACACAGU  857-875 35 UUUGCUCUGGUGAAUUACA  877-895 36AGCGUUUAGGCAUGUUUAA  998-1016 37 GCGUUUAGGCAUGUUUAAC  999-1017 38UUAGGCAUGUUUAACAUCC 1003-1021 39 UGGGUGCUGCUGAUGAAAU 1045-1063 40UGCCACCGCCAUCUUCUUC 1074-1092 41 CCUGGAAAAUGAACUCACC 1119-1137 42CGAUAUCAUCACCAAGUUC 1140-1158 43 ACCAAGUUCCUGGAAAAUG 1150-1168 44UCCAUUACUGGAACCUAUG 1207-1225 45 CCAUUACUGGAACCUAUGA 1208-1226 46ACUGGAACCUAUGAUCUGA 1213-1231 47 GGAACCUAUGAUCUGAAGA 1216-1234 48GAACCUAUGAUCUGAAGAG 1217-1235 49 CAGCAAUGGGGCUGACCUC 1269-1287 50GCAAUGGGGCUGACCUCUC 1271-1289 51 AGAGGAGGCACCCCUGAAG 1299-1317 52AGGCACCCCUGAAGCUCUC 1304-1322 53 UCUCCAAGGCCGUGCAUAA 1319-1337 54UCCAAGGCCGUGCAUAAGG 1321-1339 55 CCAAGGCCGUGCAUAAGGC 1322-1340 56CAAGGCCGUGCAUAAGGCU 1323-1341 57 AAGGCUGUGCUGACCAUCG 1336-1354 58GGCUGUGCUGACCAUCGAC 1338-1356 59 CUGCUGGGGCCAUGUUUUU 1373-1391 60GCUGGGGCCAUGUUUUUAG 1375-1393 61 CUGGGGCCAUGUUUUUAGA 1376-1394 62GGGGCCAUGUUUUUAGAGG 1378-1396 63 GGGCCAUGUUUUUAGAGGC 1379-1397 64GAGGCCAUACCCAUGUCUA 1393-1411 65 GGCCAUACCCAUGUCUAUC 1395-1413 66CCCGAGGUCAAGUUCAACA 1417-1435 67 AGGUCAAGUUCAACAAACC 1421-1439 68CAAGUUCAACAAACCCUUU 1425-1443 69 AGUUCAACAAACCCUUUGU 1427-1445 70GUUCAACAAACCCUUUGUC 1428-1446 71 UCAACAAACCCUUUGUCUU 1430-1448 72ACCCUUUGUCUUCUUAAUG 1437-1455 73 CCUUUGUCUUCUUAAUGAU 1439-1457 74UACCAAGUCUCCCCUCUUC 1467-1485 75 AAGUCUCCCCUCUUCAUGG 1471-1489 76AGUCUCCCCUCUUCAUGGG 1472-1490 77 UCUCCCCUCUUCAUGGGAA 1474-1492 78CUCCCCUCUUCAUGGGAAA 1475-1493 79 AUGACAUUAAAGAAGGGUU 1569-1587

In some embodiments, an AAT RNAi agent includes an antisense strandwherein position 19 of the antisense strand (5′→3′) is capable offorming a base pair with position 1 of a 19-mer target sequencedisclosed in Table 1. In some embodiments, an AAT RNAi agent includes anantisense strand wherein position 1 of the antisense strand (5′→3′) iscapable of forming a base pair with position 19 of the 19-mer targetsequence disclosed in Table 1.

In some embodiments, an AAT RNAi agent includes an antisense strandwherein position 2 of the antisense strand (5′→3′) is capable of forminga base pair with position 18 of the 19-mer target sequence disclosed inTable 1. In some embodiments, an AAT RNAi agent includes an antisensestrand wherein positions 2 through 18 of the antisense strand (5′→3′)are capable of forming base pairs with each of the respectivecomplementary bases located at positions 18 through 2 of the 19-mertarget sequence disclosed in Table 1.

For the RNAi agents disclosed herein, the nucleotide at position 1 ofthe antisense strand (from 5′ end→3′ end) can be perfectly complementaryto the AAT gene, or can be non-complementary to the AAT gene. In someembodiments, the nucleotide at position 1 of the antisense strand (from5′ end→3′ end) is a U, A, or dT. In some embodiments, the nucleotide atposition 1 of the antisense strand (from 5′ end→3′ end) forms an A:U orU:A base pair with the sense strand.

In some embodiments, an AAT RNAi agent antisense strand comprises thesequence of nucleotides (from 5′ end→3′ end) 2-18 or 2-19 of any of theantisense strand sequences in Table 2, Table 3, or Table 4. In someembodiments, an AAT RNAi sense strand comprises the sequence ofnucleotides (from 5′ end→3′ end) 1-17, 1-18, or 2-18 of any of the sensestrand sequences in Table 2, Table 3, or Table 5.

In some embodiments, an AAT RNAi agent is comprised of (i) an antisensestrand comprising the sequence of nucleotides (from 5′ end→3′ end) 2-18or 2-19 of any of the antisense strand sequences in Table 2, Table 3, orTable 4, and (ii) a sense strand comprising the sequence of nucleotides(from 5′ end→3′ end) 1-17 or 1-18 of any of the sense strand sequencesin Table 2, Table 3, or Table 5.

In some embodiments, the AAT RNAi agents include core 19-mer nucleotidesequences shown in the following Table 2.

TABLE 2 Example AAT RNAi Agent Antisense Strandand Sense Strand Core Stretch Base Sequences (N = any nucleobase)Antisense Sense Gene SEQ Base Sequence SEQ Base Sequence Position ID(5′ → 3′) ID (5′ → 3′) of SEQ  NO: (19-mers) NO: (19-mers) ID NO: 1  80UGUUAAACAUGCCUAAACG 429 CGUUUAGGCAUGUUUAACA 1000-1018  81AGUUAAACAUGCCUAAACG 430 CGUUUAGGCAUGUUUAACU 1000-1018  82NGUUAAACAUGCCUAAACG 431 CGUUUAGGCAUGUUUAACN 1000-1018  83NGUUAAACAUGCCUAAACN 432 NGUUUAGGCAUGUUUAACN 1000-1018  84AGAAGAUAUUGGUGCUGUU 433 AACAGCACCAAUAUCUUCU 469-487  85UGAAGAUAUUGGUGCUGUU 434 AACAGCACCAAUAUCUUCA 469-487  86NGAAGAUAUUGGUGCUGUU 435 AACAGCACCAAUAUCUUCN 469-487  87NGAAGAUAUUGGUGCUGUN 436 NACAGCACCAAUAUCUUCN 469-487  88AGGAACUUGGUGAUGAUAU 437 AUAUCAUCACCAAGUUCCU 1142-1160  89UGGAACUUGGUGAUGAUAU 438 AUAUCAUCACCAAGUUCCA 1142-1160  90NGGAACUUGGUGAUGAUAU 439 AUAUCAUCACCAAGUUCCN 1142-1160  91NGGAACUUGGUGAUGAUAN 440 NUAUCAUCACCAAGUUCCN 1142-1160  92UGUCUUCUGGGCAGCAUCU 441 AGAUGCUGCCCAGAAGACA 348-366  93AGUCUUCUGGGCAGCAUCU 442 AGAUGCUGCCCAGAAGACU 348-366  94NGUCUUCUGGGCAGCAUCU 443 AGAUGCUGCCCAGAAGACN 348-366  95NGUCUUCUGGGCAGCAUCN 444 NGAUGCUGCCCAGAAGACN 348-366  96UGUUGGACUGGUGUGCCAG 445 CUGGCACACCAGUCCAACA 454-472  97AGUUGGACUGGUGUGCCAG 446 CUGGCACACCAGUCCAACU 454-472  98NGUUGGACUGGUGUGCCAG 447 CUGGCACACCAGUCCAACN 454-472  99NGUUGGACUGGUGUGCCAN 448 NUGGCACACCAGUCCAACN 454-472 100CUGUUGGACUGGUGUGCCA 449 UGGCACACCAGUCCAACAG 455-473 101UUGUUGGACUGGUGUGCCA 450 UGGCACACCAGUCCAACAA 455-473 102AUGUUGGACUGGUGUGCCA 451 UGGCACACCAGUCCAACAU 455-473 103NUGUUGGACUGGUGUGCCA 452 UGGCACACCAGUCCAACAN 455-473 104NUGUUGGACUGGUGUGCCN 453 NGGCACACCAGUCCAACAN 455-473 105UGCUGUUGGACUGGUGUGC 454 GCACACCAGUCCAACAGCA 457-475 106AGCUGUUGGACUGGUGUGC 455 GCACACCAGUCCAACAGCU 457-475 107NGCUGUUGGACUGGUGUGC 456 GCACACCAGUCCAACAGCN 457-475 108NGCUGUUGGACUGGUGUGN 457 NCACACCAGUCCAACAGCN 457-475 109UAUUGGUGCUGUUGGACUG 458 CAGUCCAACAGCACCAAUA 463-481 110AAUUGGUGCUGUUGGACUG 459 CAGUCCAACAGCACCAAUU 463-481 111NAUUGGUGCUGUUGGACUG 460 CAGUCCAACAGCACCAAUN 463-481 112NAUUGGUGCUGUUGGACUN 461 NAGUCCAACAGCACCAAUN 463-481 113AUAUUGGUGCUGUUGGACU 462 AGUCCAACAGCACCAAUAU 464-482 114UUAUUGGUGCUGUUGGACU 463 AGUCCAACAGCACCAAUAA 464-482 115NUAUUGGUGCUGUUGGACU 464 AGUCCAACAGCACCAAUAN 464-482 116NUAUUGGUGCUGUUGGACN 465 NGUCCAACAGCACCAAUAN 464-482 117GAUAUUGGUGCUGUUGGAC 466 GUCCAACAGCACCAAUAUC 465-483 118UAUAUUGGUGCUGUUGGAC 467 GUCCAACAGCACCAAUAUA 465-483 119AAUAUUGGUGCUGUUGGAC 468 GUCCAACAGCACCAAUAUU 465-483 120NAUAUUGGUGCUGUUGGAC 469 GUCCAACAGCACCAAUAUN 465-483 121NAUAUUGGUGCUGUUGGAN 470 NUCCAACAGCACCAAUAUN 465-483 122AAGAUAUUGGUGCUGUUGG 471 CCAACAGCACCAAUAUCUU 467-485 123UAGAUAUUGGUGCUGUUGG 472 CCAACAGCACCAAUAUCUA 467-485 124NAGAUAUUGGUGCUGUUGG 473 CCAACAGCACCAAUAUCUN 467-485 125NAGAUAUUGGUGCUGUUGN 474 NCAACAGCACCAAUAUCUN 467-485 126GUAGCGAUGCUCACUGGGG 475 CCCCAGUGAGCAUCGCUAC 491-509 127UUAGCGAUGCUCACUGGGG 476 CCCCAGUGAGCAUCGCUAA 491-509 128AUAGCGAUGCUCACUGGGG 477 CCCCAGUGAGCAUCGCUAU 491-509 129NUAGCGAUGCUCACUGGGG 478 CCCCAGUGAGCAUCGCUAN 491-509 130NUAGCGAUGCUCACUGGGN 479 NCCCAGUGAGCAUCGCUAN 491-509 131AAAGGCUGUAGCGAUGCUC 480 GAGCAUCGCUACAGCCUUU 498-516 132UAAGGCUGUAGCGAUGCUC 481 GAGCAUCGCUACAGCCUUA 498-516 133NAAGGCUGUAGCGAUGCUC 482 GAGCAUCGCUACAGCCUUN 498-516 134NAAGGCUGUAGCGAUGCUN 483 NAGCAUCGCUACAGCCUUN 498-516 135GCAAAGGCUGUAGCGAUGC 484 GCAUCGCUACAGCCUUUGC 500-518 136UCAAAGGCUGUAGCGAUGC 485 GCAUCGCUACAGCCUUUGA 500-518 137ACAAAGGCUGUAGCGAUGC 486 GCAUCGCUACAGCCUUUGU 500-518 138NCAAAGGCUGUAGCGAUGC 487 GCAUCGCUACAGCCUUUGN 500-518 139NCAAAGGCUGUAGCGAUGN 488 NCAUCGCUACAGCCUUUGN 500-518 140UGCAAAGGCUGUAGCGAUG 489 CAUCGCUACAGCCUUUGCA 501-519 141AGCAAAGGCUGUAGCGAUG 490 CAUCGCUACAGCCUUUGCU 501-519 142NGCAAAGGCUGUAGCGAUG 491 NAUCGCUACAGCCUUUGCN 501-519 143NGCAAAGGCUGUAGCGAUN 492 NAUCGCUACAGCCUUUGCN 501-519 144AUUGCAAAGGCUGUAGCGA 493 UCGCUACAGCCUUUGCAAU 503-521 145UUUGCAAAGGCUGUAGCGA 494 UCGCUACAGCCUUUGCAAA 503-521 146NUUGCAAAGGCUGUAGCGA 495 UCGCUACAGCCUUUGCAAN 503-521 147NUUGCAAAGGCUGUAGCGN 496 NCGCUACAGCCUUUGCAAN 503-521 148AGCAUUGCAAAGGCUGUAG 497 CUACAGCCUUUGCAAUGCU 506-524 149UGCAUUGCAAAGGCUGUAG 498 CUACAGCCUUUGCAAUGCA 506-524 150NGCAUUGCAAAGGCUGUAG 499 CUACAGCCUUUGCAAUGCN 506-524 151NGCAUUGCAAAGGCUGUAN 500 NUACAGCCUUUGCAAUGCN 506-524 152AGAGCAUUGCAAAGGCUGU 501 ACAGCCUUUGCAAUGCUCU 508-526 153UGAGCAUUGCAAAGGCUGU 502 ACAGCCUUUGCAAUGCUCA 508-526 154NGAGCAUUGCAAAGGCUGU 503 ACAGCCUUUGCAAUGCUCN 508-526 155NGAGCAUUGCAAAGGCUGU 504 NCAGCCUUUGCAAUGCUCN 508-526 156GGAGUUCCUGGAAGCCUUC 505 GAAGGCUUCCAGGAACUCC 613-631 157UGAGUUCCUGGAAGCCUUC 506 GAAGGCUUCCAGGAACUCA 613-631 158AGAGUUCCUGGAAGCCUUC 507 GAAGGCUUCCAGGAACUCU 613-631 159NGAGUUCCUGGAAGCCUUC 508 GAAGGCUUCCAGGAACUCN 613-631 160NGAGUUCCUGGAAGCCUUN 509 NAAGGCUUCCAGGAACUCN 613-631 161UCCAAAAACUUAUCCACUA 510 UAGUGGAUAAGUUUUUGGA 710-728 162ACCAAAAACUUAUCCACUA 511 UAGUGGAUAAGUUUUUGGU 710-728 163NCCAAAAACUUAUCCACUA 512 UAGUGGAUAAGUUUUUGGN 710-728 164NCCAAAAACUUAUCCACUN 513 NAGUGGAUAAGUUUUUGGN 710-728 165AAGGCUUCUGAGUGGUACA 514 UGUACCACUCAGAAGCCUU 743-761 166UAGGCUUCUGAGUGGUACA 515 UGUACCACUCAGAAGCCUA 743-761 167NAGGCUUCUGAGUGGUACA 516 UGUACCACUCAGAAGCCUN 743-761 168NAGGCUUCUGAGUGGUACN 517 NGUACCACUCAGAAGCCUN 743-761 169GAAGGCUUCUGAGUGGUAC 518 GUACCACUCAGAAGCCUUC 744-762 170UAAGGCUUCUGAGUGGUAC 519 GUACCACUCAGAAGCCUUA 744-762 171AAAGGCUUCUGAGUGGUAC 520 GUACCACUCAGAAGCCUUU 744-762 172NAAGGCUUCUGAGUGGUAC 521 GUACCACUCAGAAGCCUUN 744-762 173NAAGGCUUCUGAGUGGUAN 522 NUACCACUCAGAAGCCUUN 744-762 174UUCUUGGCCUCUUCGGUGU 523 ACACCGAAGAGGCCAAGAA 779-797 175AUCUUGGCCUCUUCGGUGU 524 ACACCGAAGAGGCCAAGAU 779-797 176NUCUUGGCCUCUUCGGUGU 525 ACACCGAAGAGGCCAAGAN 779-797 177NUCUUGGCCUCUUCGGUGN 526 NCACCGAAGAGGCCAAGAN 779-797 178GUUUCUUGGCCUCUUCGGU 527 ACCGAAGAGGCCAAGAAAC 781-799 179UUUUCUUGGCCUCUUCGGU 528 ACCGAAGAGGCCAAGAAAA 781-799 180AUUUCUUGGCCUCUUCGGU 529 ACCGAAGAGGCCAAGAAAU 781-799 181NUUUCUUGGCCUCUUCGGU 530 ACCGAAGAGGCCAAGAAAN 781-799 182NUUUCUUGGCCUCUUCGGN 531 NCCGAAGAGGCCAAGAAAN 781-799 183UUGAUCUGUUUCUUGGCCU 532 AGGCCAAGAAACAGAUCAA 788-806 184AUGAUCUGUUUCUUGGCCU 533 AGGCCAAGAAACAGAUCAU 788-806 185NUGAUCUGUUUCUUGGCCU 534 AGGCCAAGAAACAGAUCAN 788-806 186NUGAUCUGUUUCUUGGCCN 535 NGGCCAAGAAACAGAUCAN 788-806 187GUUGAUCUGUUUCUUGGCC 536 GGCCAAGAAACAGAUCAAC 789-807 188UUUGAUCUGUUUCUUGGCC 537 GGCCAAGAAACAGAUCAAA 789-807 189AUUGAUCUGUUUCUUGGCC 538 GGCCAAGAAACAGAUCAAU 789-807 190NUUGAUCUGUUUCUUGGCC 539 GGCCAAGAAACAGAUCAAN 789-807 191NUUGAUCUGUUUCUUGGCN 540 NGCCAAGAAACAGAUCAAN 789-807 192CGUUGAUCUGUUUCUUGGC 541 GCCAAGAAACAGAUCAACG 790-808 193UGUUGAUCUGUUUCUUGGC 542 GCCAAGAAACAGAUCAACA 790-808 194AGUUGAUCUGUUUCUUGGC 543 GCCAAGAAACAGAUCAACU 790-808 195NGUUGAUCUGUUUCUUGGC 544 GCCAAGAAACAGAUCAACN 790-808 196NGUUGAUCUGUUUCUUGGN 545 NCCAAGAAACAGAUCAACN 790-808 197CACAAUUUUCCCUUGAGUA 546 UACUCAAGGGAAAAUUGUG 825-843 198UACAAUUUUCCCUUGAGUA 547 UACUCAAGGGAAAAUUGUA 825-843 199AACAAUUUUCCCUUGAGUA 548 UACUCAAGGGAAAAUUGUU 825-843 200NACAAUUUUCCCUUGAGUA 549 UACUCAAGGGAAAAUUGUN 825-843 201NACAAUUUUCCCUUGAGUN 550 NACUCAAGGGAAAAUUGUN 825-843 202UCCACAAUUUUCCCUUGAG 551 CUCAAGGGAAAAUUGUGGA 827-845 203ACCACAAUUUUCCCUUGAG 552 CUCAAGGGAAAAUUGUGGU 827-845 204NCCACAAUUUUCCCUUGAG 553 CUCAAGGGAAAAUUGUGGN 827-845 205NCCACAAUUUUCCCUUGAN 554 NUCAAGGGAAAAUUGUGGN 827-845 206AUCCACAAUUUUCCCUUGA 555 UCAAGGGAAAAUUGUGGAU 828-846 207UUCCACAAUUUUCCCUUGA 556 UCAAGGGAAAAUUGUGGAA 828-846 208NUCCACAAUUUUCCCUUGA 557 UCAAGGGAAAAUUGUGGAN 828-846 209NUCCACAAUUUUCCCUUGN 558 NCAAGGGAAAAUUGUGGAN 828-846 210UGUCAAGCUCCUUGACCAA 559 UUGGUCAAGGAGCUUGACA 847-865 211AGUCAAGCUCCUUGACCAA 560 UUGGUCAAGGAGCUUGACU 847-865 212NGUCAAGCUCCUUGACCAA 561 NUGGUCAAGGAGCUUGACA 847-865 213NGUCAAGCUCCUUGACCAA 562 NUGGUCAAGGAGCUUGACN 847-865 214GUGUCUCUGUCAAGCUCCU 563 AGGAGCUUGACAGAGACAC 854-872 215UUGUCUCUGUCAAGCUCCU 564 AGGAGCUUGACAGAGACAA 854-872 216AUGUCUCUGUCAAGCUCCU 565 AGGAGCUUGACAGAGACAU 854-872 217NUGUCUCUGUCAAGCUCCU 566 AGGAGCUUGACAGAGACAN 854-872 218NUGUCUCUGUCAAGCUCCN 567 NGGAGCUUGACAGAGACAN 854-872 219ACUGUGUCUCUGUCAAGCU 568 AGCUUGACAGAGACACAGU 857-875 220UCUGUGUCUCUGUCAAGCU 569 AGCUUGACAGAGACACAGA 857-875 221NCUGUGUCUCUGUCAAGCU 570 AGCUUGACAGAGACACAGN 857-875 222NCUGUGUCUCUGUCAAGCN 571 NGCUUGACAGAGACACAGN 857-875 223UGUAAUUCACCAGAGCAAA 572 UUUGCUCUGGUGAAUUACA 877-895 224AGUAAUUCACCAGAGCAAA 573 UUUGCUCUGGUGAAUUACU 877-895 225NGUAAUUCACCAGAGCAAA 574 UUUGCUCUGGUGAAUUACN 877-895 226NGUAAUUCACCAGAGCAAN 575 NUUGCUCUGGUGAAUUACN 877-895 227UUAAACAUGCCUAAACGCU 576 AGCGUUUAGGCAUGUUUAA  998-1016 228AUAAACAUGCCUAAACGCU 577 AGCGUUUAGGCAUGUUUAU  998-1016 229NUAAACAUGCCUAAACGCU 578 AGCGUUUAGGCAUGUUUAN  998-1016 230NUAAACAUGCCUAAACGCN 579 NGCGUUUAGGCAUGUUUAN  998-1016 231GUUAAACAUGCCUAAACGC 580 GCGUUUAGGCAUGUUUAAC  999-1017 232UUUAAACAUGCCUAAACGC 581 GCGUUUAGGCAUGUUUAAA  999-1017 233AUUAAACAUGCCUAAACGC 582 GCGUUUAGGCAUGUUUAAU  999-1017 234NUUAAACAUGCCUAAACGC 583 GCGUUUAGGCAUGUUUAAN  999-1017 235NUUAAACAUGCCUAAACGN 584 NCGUUUAGGCAUGUUUAAN  999-1017 236GGAUGUUAAACAUGCCUAA 585 UUAGGCAUGUUUAACAUCC 1003-1021 237UGAUGUUAAACAUGCCUAA 586 UUAGGCAUGUUUAACAUCA 1003-1021 238AGAUGUUAAACAUGCCUAA 587 UUAGGCAUGUUUAACAUCU 1003-1021 239NGAUGUUAAACAUGCCUAA 588 UUAGGCAUGUUUAACAUCN 1003-1021 240NGAUGUUAAACAUGCCUAN 589 NUAGGCAUGUUUAACAUCN 1003-1021 241AUUUCAUCAGCAGCACCCA 590 UGGGUGCUGCUGAUGAAAU 1045-1063 242UUUUCAUCAGCAGCACCCA 591 UGGGUGCUGCUGAUGAAAA 1045-1063 243NUUUCAUCAGCAGCACCCA 592 UGGGUGCUGCUGAUGAAAN 1045-1063 244NUUUCAUCAGCAGCACCCN 593 NGGGUGCUGCUGAUGAAAN 1045-1063 245GAAGAAGAUGGCGGUGGCA 594 UGCCACCGCCAUCUUCUUC 1074-1092 246UAAGAAGAUGGCGGUGGCA 595 UGCCACCGCCAUCUUCUUA 1074-1092 247AAAGAAGAUGGCGGUGGCA 596 UGCCACCGCCAUCUUCUUU 1074-1092 248NAAGAAGAUGGCGGUGGCA 597 UGCCACCGCCAUCUUCUUN 1074-1092 249NAAGAAGAUGGCGGUGGCN 598 NGCCACCGCCAUCUUCUUN 1074-1092 250GGUGAGUUCAUUUUCCAGG 599 CCUGGAAAAUGAACUCACC 1119-1137 251UGUGAGUUCAUUUUCCAGG 600 CCUGGAAAAUGAACUCACA 1119-1137 252AGUGAGUUCAUUUUCCAGG 601 CCUGGAAAAUGAACUCACU 1119-1137 253NGUGAGUUCAUUUUCCAGG 602 CCUGGAAAAUGAACUCACN 1119-1137 254NGUGAGUUCAUUUUCCAGN 603 NCUGGAAAAUGAACUCACN 1119-1137 255GAACUUGGUGAUGAUAUCG 604 CGAUAUCAUCACCAAGUUC 1140-1158 256UAACUUGGUGAUGAUAUCG 605 CGAUAUCAUCACCAAGUUA 1140-1158 257AAACUUGGUGAUGAUAUCG 606 CGAUAUCAUCACCAAGUUU 1140-1158 258NAACUUGGUGAUGAUAUCG 607 CGAUAUCAUCACCAAGUUN 1140-1158 259NAACUUGGUGAUGAUAUCN 608 NGAUAUCAUCACCAAGUUN 1140-1158 260CAUUUUCCAGGAACUUGGU 609 ACCAAGUUCCUGGAAAAUG 1150-1168 261UAUUUUCCAGGAACUUGGU 610 ACCAAGUUCCUGGAAAAUA 1150-1168 262AAUUUUCCAGGAACUUGGU 611 ACCAAGUUCCUGGAAAAUU 1150-1168 263NAUUUUCCAGGAACUUGGU 612 ACCAAGUUCCUGGAAAAUN 1150-1168 264NAUUUUCCAGGAACUUGGN 613 NCCAAGUUCCUGGAAAAUN 1150-1168 265CAUAGGUUCCAGUAAUGGA 614 UCCAUUACUGGAACCUAUG 1207-1225 266UAUAGGUUCCAGUAAUGGA 615 UCCAUUACUGGAACCUAUA 1207-1225 267AAUAGGUUCCAGUAAUGGA 616 UCCAUUACUGGAACCUAUU 1207-1225 268NAUAGGUUCCAGUAAUGGA 617 UCCAUUACUGGAACCUAUN 1207-1225 269NAUAGGUUCCAGUAAUGGN 618 NCCAUUACUGGAACCUAUN 1207-1225 270UCAUAGGUUCCAGUAAUGG 619 CCAUUACUGGAACCUAUGA 1208-1226 271ACAUAGGUUCCAGUAAUGG 620 CCAUUACUGGAACCUAUGU 1208-1226 272NCAUAGGUUCCAGUAAUGG 621 CCAUUACUGGAACCUAUGN 1208-1226 273NCAUAGGUUCCAGUAAUGN 622 NCAUUACUGGAACCUAUGN 1208-1226 274UCAGAUCAUAGGUUCCAGU 623 ACUGGAACCUAUGAUCUGA 1213-1231 275ACAGAUCAUAGGUUCCAGU 624 ACUGGAACCUAUGAUCUGU 1213-1231 276NCAGAUCAUAGGUUCCAGU 625 ACUGGAACCUAUGAUCUGN 1213-1231 277NCAGAUCAUAGGUUCCAGN 626 NCUGGAACCUAUGAUCUGN 1213-1231 278UCUUCAGAUCAUAGGUUCC 627 GGAACCUAUGAUCUGAAGA 1216-1234 279ACUUCAGAUCAUAGGUUCC 628 GGAACCUAUGAUCUGAAGU 1216-1234 280NCUUCAGAUCAUAGGUUCC 629 GGAACCUAUGAUCUGAAGN 1216-1234 281NCUUCAGAUCAUAGGUUCN 630 NGAACCUAUGAUCUGAAGN 1216-1234 282CUCUUCAGAUCAUAGGUUC 631 GAACCUAUGAUCUGAAGAG 1217-1235 283UUCUUCAGAUCAUAGGUUC 632 GAACCUAUGAUCUGAAGAA 1217-1235 284AUCUUCAGAUCAUAGGUUC 633 GAACCUAUGAUCUGAAGAU 1217-1235 285NUCUUCAGAUCAUAGGUUC 634 GAACCUAUGAUCUGAAGAG 1217-1235 286NUCUUCAGAUCAUAGGUUN 635 NAACCUAUGAUCUGAAGAN 1217-1235 287GAGGUCAGCCCCAUUGCUG 636 CAGCAAUGGGGCUGACCUC 1269-1287 288UAGGUCAGCCCCAUUGCUG 637 CAGCAAUGGGGCUGACCUA 1269-1287 289AAGGUCAGCCCCAUUGCUG 638 CAGCAAUGGGGCUGACCUU 1269-1287 290NAGGUCAGCCCCAUUGCUG 639 CAGCAAUGGGGCUGACCUN 1269-1287 291NAGGUCAGCCCCAUUGCUN 640 NAGCAAUGGGGCUGACCUN 1269-1287 292GAGAGGUCAGCCCCAUUGC 641 GCAAUGGGGCUGACCUCUC 1271-1289 293UAGAGGUCAGCCCCAUUGC 642 GCAAUGGGGCUGACCUCUA 1271-1289 294AAGAGGUCAGCCCCAUUGC 643 GCAAUGGGGCUGACCUCUU 1271-1289 295NAGAGGUCAGCCCCAUUGC 644 GCAAUGGGGCUGACCUCUN 1271-1289 296NAGAGGUCAGCCCCAUUGN 645 NCAAUGGGGCUGACCUCUN 1271-1289 297CUUCAGGGGUGCCUCCUCU 646 AGAGGAGGCACCCCUGAAG 1299-1317 298UUUCAGGGGUGCCUCCUCU 647 AGAGGAGGCACCCCUGAAA 1299-1317 299AUUCAGGGGUGCCUCCUCU 648 AGAGGAGGCACCCCUGAAU 1299-1317 300NUUCAGGGGUGCCUCCUCU 649 AGAGGAGGCACCCCUGAAN 1299-1317 301NUUCAGGGGUGCCUCCUCN 650 NGAGGAGGCACCCCUGAAN 1299-1317 302GAGAGCUUCAGGGGUGCCU 651 AGGCACCCCUGAAGCUCUC 1304-1322 303UAGAGCUUCAGGGGUGCCU 652 AGGCACCCCUGAAGCUCUA 1304-1322 304AAGAGCUUCAGGGGUGCCU 653 AGGCACCCCUGAAGCUCUU 1304-1322 305NAGAGCUUCAGGGGUGCCU 654 AGGCACCCCUGAAGCUCUN 1304-1322 306NAGAGCUUCAGGGGUGCCN 655 NGGCACCCCUGAAGCUCUN 1304-1322 307UUAUGCACGGCCUUGGAGA 656 UCUCCAAGGCCGUGCAUAA 1319-1337 308AUAUGCACGGCCUUGGAGA 657 UCUCCAAGGCCGUGCAUAU 1319-1337 309NUAUGCACGGCCUUGGAGA 658 UCUCCAAGGCCGUGCAUAN 1319-1337 310NUAUGCACGGCCUUGGAGN 659 NCUCCAAGGCCGUGCAUAN 1319-1337 311CCUUAUGCACGGCCUUGGA 660 UCCAAGGCCGUGCAUAAGG 1321-1339 312UCUUAUGCACGGCCUUGGA 661 UCCAAGGCCGUGCAUAAGA 1321-1339 313ACUUAUGCACGGCCUUGGA 662 UCCAAGGCCGUGCAUAAGU 1321-1339 314NCUUAUGCACGGCCUUGGA 663 UCCAAGGCCGUGCAUAAGN 1321-1339 315NCUUAUGCACGGCCUUGGN 664 NCCAAGGCCGUGCAUAAGN 1321-1339 316GCCUUAUGCACGGCCUUGG 665 CCAAGGCCGUGCAUAAGGC 1322-1340 317UCCUUAUGCACGGCCUUGG 666 CCAAGGCCGUGCAUAAGGA 1322-1340 318ACCUUAUGCACGGCCUUGG 667 CCAAGGCCGUGCAUAAGGU 1322-1340 319NCCUUAUGCACGGCCUUGG 668 CCAAGGCCGUGCAUAAGGN 1322-1340 320NCCUUAUGCACGGCCUUGN 669 NCAAGGCCGUGCAUAAGGN 1322-1340 321AGCCUUAUGCACGGCCUUG 670 CAAGGCCGUGCAUAAGGCU 1323-1341 322UGCCUUAUGCACGGCCUUG 671 CAAGGCCGUGCAUAAGGCA 1323-1341 323NGCCUUAUGCACGGCCUUG 672 CAAGGCCGUGCAUAAGGCN 1323-1341 324NGCCUUAUGCACGGCCUUN 673 NAAGGCCGUGCAUAAGGCN 1323-1341 325CGAUGGUCAGCACAGCCUU 674 AAGGCUGUGCUGACCAUCG 1336-1354 326UGAUGGUCAGCACAGCCUU 675 AAGGCUGUGCUGACCAUCA 1336-1354 327AGAUGGUCAGCACAGCCUU 676 AAGGCUGUGCUGACCAUCU 1336-1354 328NGAUGGUCAGCACAGCCUU 677 AAGGCUGUGCUGACCAUCN 1336-1354 329NGAUGGUCAGCACAGCCUN 678 NAGGCUGUGCUGACCAUCN 1336-1354 330GUCGAUGGUCAGCACAGCC 679 GGCUGUGCUGACCAUCGAC 1338-1356 331UUCGAUGGUCAGCACAGCC 680 GGCUGUGCUGACCAUCGAA 1338-1356 332AUCGAUGGUCAGCACAGCC 681 GGCUGUGCUGACCAUCGAU 1338-1356 333NUCGAUGGUCAGCACAGCC 682 GGCUGUGCUGACCAUCGAN 1338-1356 334NUCGAUGGUCAGCACAGCN 683 NGCUGUGCUGACCAUCGAN 1338-1356 335AAAAACAUGGCCCCAGCAG 684 CUGCUGGGGCCAUGUUUUU 1373-1391 336UAAAACAUGGCCCCAGCAG 685 CUGCUGGGGCCAUGUUUUA 1373-1391 337NAAAACAUGGCCCCAGCAG 686 CUGCUGGGGCCAUGUUUUN 1373-1391 338NAAAACAUGGCCCCAGCAN 687 NUGCUGGGGCCAUGUUUUN 1373-1391 339CUAAAAACAUGGCCCCAGC 688 GCUGGGGCCAUGUUUUUAG 1375-1393 340UUAAAAACAUGGCCCCAGC 689 GCUGGGGCCAUGUUUUUAA 1375-1393 341AUAAAAACAUGGCCCCAGC 690 GCUGGGGCCAUGUUUUUAU 1375-1393 342NUAAAAACAUGGCCCCAGC 691 GCUGGGGCCAUGUUUUUAN 1375-1393 343NUAAAAACAUGGCCCCAGN 692 NCUGGGGCCAUGUUUUUAN 1375-1393 344UCUAAAAACAUGGCCCCAG 693 CUGGGGCCAUGUUUUUAGA 1376-1394 345ACUAAAAACAUGGCCCCAG 694 CUGGGGCCAUGUUUUUAGU 1376-1394 346NCUAAAAACAUGGCCCCAG 695 CUGGGGCCAUGUUUUUAGN 1376-1394 347NCUAAAAACAUGGCCCCAN 696 NUGGGGCCAUGUUUUUAGN 1376-1394 348CCUCUAAAAACAUGGCCCC 697 GGGGCCAUGUUUUUAGAGG 1378-1396 349UCUCUAAAAACAUGGCCCC 698 GGGGCCAUGUUUUUAGAGA 1378-1396 350ACUCUAAAAACAUGGCCCC 699 GGGGCCAUGUUUUUAGAGU 1378-1396 351NCUCUAAAAACAUGGCCCC 700 GGGGCCAUGUUUUUAGAGN 1378-1396 352NCUCUAAAAACAUGGCCCN 701 NGGGCCAUGUUUUUAGAGN 1378-1396 353GCCUCUAAAAACAUGGCCC 702 GGGCCAUGUUUUUAGAGGC 1379-1397 354UCCUCUAAAAACAUGGCCC 703 GGGCCAUGUUUUUAGAGGA 1379-1397 355ACCUCUAAAAACAUGGCCC 704 GGGCCAUGUUUUUAGAGGU 1379-1397 356NCCUCUAAAAACAUGGCCC 705 GGGCCAUGUUUUUAGAGGN 1379-1397 357NCCUCUAAAAACAUGGCCN 706 NGGCCAUGUUUUUAGAGGN 1379-1397 358UAGACAUGGGUAUGGCCUC 707 GAGGCCAUACCCAUGUCUA 1393-1411 359AAGACAUGGGUAUGGCCUC 708 GAGGCCAUACCCAUGUCUU 1393-1411 360NAGACAUGGGUAUGGCCUC 709 GAGGCCAUACCCAUGUCUN 1393-1411 361NAGACAUGGGUAUGGCCUN 710 NAGGCCAUACCCAUGUCUN 1393-1411 362GAUAGACAUGGGUAUGGCC 711 GGCCAUACCCAUGUCUAUC 1395-1413 363UAUAGACAUGGGUAUGGCC 712 GGCCAUACCCAUGUCUAUA 1395-1413 364AAUAGACAUGGGUAUGGCC 713 GGCCAUACCCAUGUCUAUU 1395-1413 365NAUAGACAUGGGUAUGGCC 714 GGCCAUACCCAUGUCUAUN 1395-1413 366NAUAGACAUGGGUAUGGCN 715 NGCCAUACCCAUGUCUAUN 1395-1413 367UGUUGAACUUGACCUCGGG 716 CCCGAGGUCAAGUUCAACA 1417-1435 368AGUUGAACUUGACCUCGGG 717 CCCGAGGUCAAGUUCAACU 1417-1435 369NGUUGAACUUGACCUCGGG 718 CCCGAGGUCAAGUUCAACN 1417-1435 370NGUUGAACUUGACCUCGGN 719 NCCGAGGUCAAGUUCAACN 1417-1435 371GGUUUGUUGAACUUGACCU 720 AGGUCAAGUUCAACAAACC 1421-1439 372UGUUUGUUGAACUUGACCU 721 AGGUCAAGUUCAACAAACA 1421-1439 373AGUUUGUUGAACUUGACCU 722 AGGUCAAGUUCAACAAACU 1421-1439 374NGUUUGUUGAACUUGACCU 723 AGGUCAAGUUCAACAAACN 1421-1439 375NGUUUGUUGAACUUGACCN 724 NGGUCAAGUUCAACAAACN 1421-1439 376AAAGGGUUUGUUGAACUUG 725 CAAGUUCAACAAACCCUUU 1425-1443 377UAAGGGUUUGUUGAACUUG 726 CAAGUUCAACAAACCCUUA 1425-1443 378NAAGGGUUUGUUGAACUUG 727 CAAGUUCAACAAACCCUUN 1425-1443 379NAAGGGUUUGUUGAACUUN 728 NAAGUUCAACAAACCCUUN 1425-1443 380ACAAAGGGUUUGUUGAACU 729 AGUUCAACAAACCCUUUGU 1427-1445 381UCAAAGGGUUUGUUGAACU 730 AGUUCAACAAACCCUUUGA 1427-1445 382NCAAAGGGUUUGUUGAACU 731 AGUUCAACAAACCCUUUGN 1427-1445 383NCAAAGGGUUUGUUGAACN 732 NGUUCAACAAACCCUUUGN 1427-1445 384GACAAAGGGUUUGUUGAAC 733 GUUCAACAAACCCUUUGUC 1428-1446 385UACAAAGGGUUUGUUGAAC 734 GUUCAACAAACCCUUUGUA 1428-1446 386AACAAAGGGUUUGUUGAAC 735 GUUCAACAAACCCUUUGUU 1428-1446 387NACAAAGGGUUUGUUGAAC 736 GUUCAACAAACCCUUUGUN 1428-1446 388NACAAAGGGUUUGUUGAAN 737 NUUCAACAAACCCUUUGUN 1428-1446 389AAGACAAAGGGUUUGUUGA 738 UCAACAAACCCUUUGUCUU 1430-1448 390UAGACAAAGGGUUUGUUGA 739 UCAACAAACCCUUUGUCUA 1430-1448 391NAGACAAAGGGUUUGUUGA 740 UCAACAAACCCUUUGUCUN 1430-1448 392NAGACAAAGGGUUUGUUGN 741 NCAACAAACCCUUUGUCUN 1430-1448 393CAUUAAGAAGACAAAGGGU 742 ACCCUUUGUCUUCUUAAUG 1437-1455 394UAUUAAGAAGACAAAGGGU 743 ACCCUUUGUCUUCUUAAUA 1437-1455 395AAUUAAGAAGACAAAGGGU 744 ACCCUUUGUCUUCUUAAUU 1437-1455 396NAUUAAGAAGACAAAGGGU 745 ACCCUUUGUCUUCUUAAUN 1437-1455 397NAUUAAGAAGACAAAGGGN 746 NCCCUUUGUCUUCUUAAUN 1437-1455 398AUCAUUAAGAAGACAAAGG 747 CCUUUGUCUUCUUAAUGAU 1439-1457 399UUCAUUAAGAAGACAAAGG 748 CCUUUGUCUUCUUAAUGAA 1439-1457 400NUCAUUAAGAAGACAAAGG 749 CCUUUGUCUUCUUAAUGAN 1439-1457 401NUCAUUAAGAAGACAAAGN 750 NCUUUGUCUUCUUAAUGAN 1439-1457 402GAAGAGGGGAGACUUGGUA 751 UACCAAGUCUCCCCUCUUC 1467-1485 403UAAGAGGGGAGACUUGGUA 752 UACCAAGUCUCCCCUCUUA 1467-1485 404AAAGAGGGGAGACUUGGUA 753 UACCAAGUCUCCCCUCUUU 1467-1485 405NAAGAGGGGAGACUUGGUA 754 UACCAAGUCUCCCCUCUUN 1467-1485 406NAAGAGGGGAGACUUGGUN 755 NACCAAGUCUCCCCUCUUN 1467-1485 407CCAUGAAGAGGGGAGACUU 756 AAGUCUCCCCUCUUCAUGG 1471-1489 408UCAUGAAGAGGGGAGACUU 757 AAGUCUCCCCUCUUCAUGA 1471-1489 409ACAUGAAGAGGGGAGACUU 758 AAGUCUCCCCUCUUCAUGU 1471-1489 410NCAUGAAGAGGGGAGACUU 759 AAGUCUCCCCUCUUCAUGN 1471-1489 411NCAUGAAGAGGGGAGACUN 760 NAGUCUCCCCUCUUCAUGN 1471-1489 412CCCAUGAAGAGGGGAGACU 761 AGUCUCCCCUCUUCAUGGG 1472-1490 413UCCAUGAAGAGGGGAGACU 762 AGUCUCCCCUCUUCAUGGA 1472-1490 414ACCAUGAAGAGGGGAGACU 763 AGUCUCCCCUCUUCAUGGU 1472-1490 415NCCAUGAAGAGGGGAGACU 764 AGUCUCCCCUCUUCAUGGN 1472-1490 416NCCAUGAAGAGGGGAGACN 765 NGUCUCCCCUCUUCAUGGN 1472-1490 417UUCCCAUGAAGAGGGGAGA 766 UCUCCCCUCUUCAUGGGAA 1474-1492 418AUCCCAUGAAGAGGGGAGA 767 UCUCCCCUCUUCAUGGGAU 1474-1492 419NUCCCAUGAAGAGGGGAGA 768 UCUCCCCUCUUCAUGGGAN 1474-1492 420NUCCCAUGAAGAGGGGAGN 769 NCUCCCCUCUUCAUGGGAN 1474-1492 421UUUCCCAUGAAGAGGGGAG 770 CUCCCCUCUUCAUGGGAAA 1475-1493 422AUUCCCAUGAAGAGGGGAG 771 CUCCCCUCUUCAUGGGAAU 1475-1493 423NUUCCCAUGAAGAGGGGAG 772 CUCCCCUCUUCAUGGGAAN 1475-1493 424NUUCCCAUGAAGAGGGGAN 773 NUCCCCUCUUCAUGGGAAN 1475-1493 425AACCCUUCUUUAAUGUCAU 774 AUGACAUUAAAGAAGGGUU 1569-1587 426UACCCUUCUUUAAUGUCAU 775 AUGACAUUAAAGAAGGGUA 1569-1587 427NACCCUUCUUUAAUGUCAU 776 AUGACAUUAAAGAAGGGUN 1569-1587 428NACCCUUCUUUAAUGUCAN 777 NUGACAUUAAAGAAGGGUN 1569-1587

TABLE 3 Example AAT RNAi Agent Antisense Strandand Sense Strand Base Sequences SEQ Antisense SEQ Sense ID Base SequenceID Base Sequence NO: (5′ → 3′) NO: (5′ → 3′) 778 GGAACUUGGUGAUGAUAU  840AUAUCAUCACCAAGUUCC 779 GAUCAUAGGUUCCAGUAA  841 UUACUGGAACCUAUGAUC 780ACAGCCUUAUGCACGGCC  842 GGCCGUGCAUAAGGCUGU 781 UCGAUGGUCAGCACAGCC  843GGCUGUGCUGACCAUCGA 782 CAAAGGGUUUGUUGAACU  844 AGUUCAACAAACCCUUUG 783TGGAACUUGGUGAUGAUAUTT  845 UAUAUAUCAUCACCAAGUUCCAT 783TGGAACUUGGUGAUGAUAUTT  846 AUAUCAUCACCAAGUUCCAT 784TGGAACUUGGUGAUGAUAUCGUG  847 CGAUAUCAUCACCAAGUUCCA 785 ACUUGGUGAUGAUAUTT 848 UAUCAUCACCAAGUUCCAT 786 TGGAACTTGGTGATGATATTT  849TATATATCATCACCAAGTTCCAT 787 UUUAAACAUGCCUAAACGCUU  850GCGUUUAGGCAUGUUUAAAUU 788 UGCAUUGCCCAGGUAUUUCUU  851GAAAUACCUGGGCAAUGCAUU 789 UGGAACUUGGUGAUGAUAUUU  852AUAUCAUCACCAAGUUCCAUU 790 UGAUCAUAGGUUCCAGUAAUU  853UUACUGGAACCUAUGAUCAUU 791 UACAGCCUUAUGCACGGCCUU  854GGCCGUGCAUAAGGCUGUAUU 792 UUCGAUGGUCAGCACAGCCUU  855GGCUGUGCUGACCAUCGAAUU 793 UCAAAGGGUUUGUUGAACUUU  856AGUUCAACAAACCCUUUGAUU 794 UGUUAAACAUGCCUAAACGUU  857CGUUUAGGCAUGUUUAACAUU 795 UUUAAACGUGCCUAAACGCUG  858CAGCGUUUAGGCAUGUUUAAA 796 UGCAUUGCCCAGGUAUUUCAG  859CUGAAAUACCUGGGCAAUGCA 797 UGGAACUUGGUGAUGAUAUCG  847CGAUAUCAUCACCAAGUUCCA 798 UGAUCAUAGGUUCCAGUAAUG  860CAUUACUGGAACCUAUGAUCA 791 UACAGCCUUAUGCACGGCCUU  861AAGGCCGUGCAUAAGGCUGUA 792 UUCGAUGGUCAGCACAGCCUU  862AAGGCUGUGCUGACCAUCGAA 799 UCAAAGGGUUUGUUGAACUUG  863CAAGUUCAACAAACCCUUUGA 800 UGUUAAACAUGCCUAAACGCG  864CGCGUUUAGGCAUGUUUAACA 801 UGUUAAACAUGCCUAAACGCU  857CGUUUAGGCAUGUUUAACAUU 794 UGUUAAACAUGCCUAAACGUU 1265 CGUUUAGGCAUGUUUAACA801 UGUUAAACAUGCCUAAACGCU 1265 CGUUUAGGCAUGUUUAACA 794UGUUAAACAUGCCUAAACGUU  865 AACGUUUAGGCAUGUUUAACA 801UGUUAAACAUGCCUAAACGCU  866 AGCGUUUAGGCAUGUUUAACA 802UGUUAAACAUGCCUAAACGCUUC  866 AGCGUUUAGGCAUGUUUAACA 803UGCUGUUGGACUGGUGUGCUU 1266 GCACACCAGUCCAACAGCA 804 UGCUGUUGGACUGGUGUGCCA1266 GCACACCAGUCCAACAGCA 804 UGCUGUUGGACUGGUGUGCCA  867UGGCACACCAGUCCAACAGCA 803 UGCUGUUGGACUGGUGUGCUU  868AAGCACACCAGUCCAACAGCA 805 UGCUGUUGGACUGGUGUGCCAUU  867UGGCACACCAGUCCAACAGCA 806 UGCUGUUGGACUGGUGUGCCAGC  867UGGCACACCAGUCCAACAGCA 807 UAAGGCUUCUGAGUGGUACUU 1267 GUACCACUCAGAAGCCUUA808 UAAGGCUUCUGAGUGGUACAA 1267 GUACCACUCAGAAGCCUUA 808UAAGGCUUCUGAGUGGUACAA  869 UUGUACCACUCAGAAGCCUUA 809UAAGGCUUCUGAGUGGUACAACU  869 UUGUACCACUCAGAAGCCUUA 810GAAGGCUUCUGAGUGGUACUU 1268 GUACCACUCAGAAGCCUUC 811 AAGACAAAGGGUUUGUUGAUU1269 UCAACAAACCCUUUGUCUU 812 AAGACAAAGGGUUUGUUGAAC 1269UCAACAAACCCUUUGUCUU 812 AAGACAAAGGGUUUGUUGAAC  870 GUUCAACAAACCCUUUGUCUU813 UAGACAAAGGGUUUGUUGAAC  871 GUUCAACAAACCCUUUGUCUA 814AAGACAAAGGGUUUGUUGAACUU  870 GUUCAACAAACCCUUUGUCUU 815UAGACAUGGGUAUGGCCUCUU 1270 GAGGCCAUACCCAUGUCUA 816 UAGACAUGGGUAUGGCCUCUA1270 GAGGCCAUACCCAUGUCUA 816 UAGACAUGGGUAUGGCCUCUA  872UAGAGGCCAUACCCAUGUCUA 817 UAGACAUGGGUAUGGCCUCUAAA  872UAGAGGCCAUACCCAUGUCUA 818 UAGACAUGGGUAUGGCCUCUAUU  872UAGAGGCCAUACCCAUGUCUA 819 UUUGAUCUGUUUCUUGGCCUU 1271 GGCCAAGAAACAGAUCAAA820 UUUGAUCUGUUUCUUGGCCUC 1271 GGCCAAGAAACAGAUCAAA 820UUUGAUCUGUUUCUUGGCCUC  873 GAGGCCAAGAAACAGAUCAAA 821UUUGAUCUGUUUCUUGGCCUCUU  873 GAGGCCAAGAAACAGAUCAAA 822UGUUGGACUGGUGUGCCAGUU 1272 CUGGCACACCAGUCCAACA 823 UGUUGGACUGGUGUGCCAGCU 874 AGCUGGCACACCAGUCCAACA 824 UGUUGGACUGGUGUGCCAGCUGG  874AGCUGGCACACCAGUCCAACA 825 UGUUGGACUGGUGUGCCAGCUG  875GCUGGCACACCAGUCCAACA 826 AAAGGGUUUGUUGAACUUGUU 1273 CAAGUUCAACAAACCCUUU827 AAAGGGUUUGUUGAACUUGAC  876 GUCAAGUUCAACAAACCCUUU 828UAAGGGUUUGUUGAACUUGACCU  877 GUCAAGUUCAACAAACCCUUA 829UAAGGGUUUGUUGAACUUGAC  877 GUCAAGUUCAACAAACCCUUA 830UAUUGGUGCUGUUGGACUGUU 1274 CAGUCCAACAGCACCAAUA 831 UAUUGGUGCUGUUGGACUGGU 878 ACCAGUCCAACAGCACCAAUA 832 UAUUGGUGCUGUUGGACUGGUU  879CCAGUCCAACAGCACCAAUA 833 UUGUUGGACUGGUGUGCCAG  880 CUGGCACACCAGUCCAACAA834 UUGUUGGACUGGUGUGCCAGCU  880 CUGGCACACCAGUCCAACAA 835UAUAGACAUGGGUAUGGCCUC 1275 GGCCAUACCCAUGUCUAUA 835 UAUAGACAUGGGUAUGGCCUC 881 GAGGCCAUACCCAUGUCUAUA 836 UCAAAGGGUUUGUUGAACUUGAC  882GUCAAGUUCAACAAACCCUUUGA 836 UCAAAGGGUUUGUUGAACUUGAC  863CAAGUUCAACAAACCCUUUGA 837 UUAUUGGUGCUGUUGGACUGG  883CCAGUCCAACAGCACCAAUAA 838 UGUUAAACAUGCCUAAACGC  884 GCGUUUAGGCAUGUUUAACA839 UGUUAAACAUGCCUAAACGCUU  884 GCGUUUAGGCAUGUUUAACA 839UGUUAAACAUGCCUAAACGCUU  885 GCGUUUAGGCAUGUUUAACAUU 800UGUUAAACAUGCCUAAACGCG  886 CGCGUUUAGGCAUGUUUAACAUU 801UGUUAAACAUGCCUAAACGCU  887 AGCGUUUAGGCAUGUUUAACAUU 838UGUUAAACAUGCCUAAACGC  885 GCGUUUAGGCAUGUUUAACAUU

The AAT RNAi agent sense strands and antisense strands that comprise orconsist of the nucleotide sequences in Table 2 or Table 3 can bemodified nucleotides or unmodified nucleotides. In some embodiments, theAAT RNAi agents having the sense and antisense strand sequences thatcomprise or consist of any of the nucleotide sequences in Table 2 orTable 3 are all or substantially all modified nucleotides.

In some embodiments, the antisense strand of an AAT RNAi agent disclosedherein differs by 0, 1, 2, or 3 nucleotides from any of the antisensestrand sequences in Table 2 or Table 3. In some embodiments, the sensestrand of an AAT RNAi agent disclosed herein differs by 0, 1, 2, or 3nucleotides from any of the sense strand sequences in Table 2 or Table3.

As used herein, each N listed in a sequence disclosed in Table 2 may beindependently selected. In some embodiments, an N nucleotide listed in asequence disclosed in Table 2 has a nucleobase that is complementary tothe N nucleotide at the corresponding position on the other strand. Insome embodiments, an N nucleotide listed in a sequence disclosed inTable 2 has a nucleobase that is not complementary to the N nucleotideat the corresponding position on the other strand. In some embodiments,an N nucleotide listed in a sequence disclosed in Table 2 has anucleobase that is the same as the N nucleotide at the correspondingposition on the other strand. In some embodiments, an N nucleotidelisted in a sequence disclosed in Table 2 has a nucleobase that isdifferent from the N nucleotide at the corresponding position on theother strand.

Certain modified AAT RNAi agent sense and antisense strands are providedin Table 4 and Table 5. Modified AAT RNAi agent antisense strands, aswell as their underlying unmodified nucleobase sequences, are providedin Table 4. Modified AAT RNAi agent sense strands, as well as theirunderlying unmodified sequences, are provided in Table 5. In forming AATRNAi agents, each of the nucleotides in each of the unmodified sequenceslisted in Tables 4 and 5, as well as in Table 2 and Table 3, above, canbe a modified nucleotide.

The AAT RNAi agents described herein are formed by annealing anantisense strand with a sense strand. A sense strand containing asequence listed in Table 2, Table 3, or Table 5, can be hybridized toany antisense strand containing a sequence listed in Table 2, Table 3,or Table 4, provided the two sequences have a region of at least 85%complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotidesequence.

In some embodiments, an AAT RNAi agent antisense strand comprises anucleotide sequence of any of the sequences in Table 2, Table 3, orTable 4.

In some embodiments, an AAT RNAi agent comprises or consists of a duplexhaving the nucleobase sequences of the sense strand and the antisensestrand of any of the sequences in Table 2 or Table 3.

Examples of antisense strands containing modified nucleotides areprovided in Table 4. Examples of sense strands containing modifiednucleotides are provided in Table 5.

As used in Tables 4 and 5, the following notations are used to indicatemodified nucleotides, targeting groups, and linking groups. As theperson of ordinary skill in the art would readily understand, unlessotherwise indicated by the sequence, that when present in anoligonucleotide, the monomers are mutually linked by5′-3′-phosphodiester bonds:

-   -   A=adenosine-3′-phosphate;    -   C=cytidine-3′-phosphate;    -   G=guanosine-3′-phosphate;    -   U=uridine-3′-phosphate    -   n=any 2′-OMe modified nucleotide    -   a=2′-O-methyladenosine-3′-phosphate    -   as =2′-O-methyladenosine-3′-phosphorothioate    -   c=2′-O-methylcytidine-3′-phosphate    -   cs=2′-O-methylcytidine-3′-phosphorothioate    -   g=2′-O-methylguanosine-3′-phosphate    -   gs=2′-O-methylguanosine-3′-phosphorothioate    -   t=2′-O-methyl-5-methyluridine-3′-phosphate    -   is =2′-O-methyl-5-methyluridine-3′-phosphorothioate    -   u=2′-O-methyluridine-3′-phosphate    -   us=2′-O-methyluridine-3′-phosphorothioate    -   Nf=any 2′-fluoro modified nucleotide    -   Af=2′-fluoroadenosine-3′-phosphate    -   Afs=2′-fluoroadenosine-3′-phosporothioate    -   Cf=2′-fluorocytidine-3′-phosphate    -   Cfs=2′-fluorocytidine-3′-phosphorothioate    -   Gf=2′-fluoroguanosine-3′-phosphate    -   Gfs=2′-fluoroguanosine-3′-phosphorothioate    -   Tf=2′-fluoro-5′-methyluridine-3′-phosphate    -   Tfs=2′-fluoro-5′-methyluridine-3′-phosphorothioate    -   Uf=2′-fluorouridine-3′-phosphate    -   Ufs=2′-fluorouridine-3′-phosphorothioate    -   dN=any 2′-deoxyribonucleotide    -   dT=2′-deoxythymidine-3′-phosphate    -   N_(UNA)=2′,3′-seco nucleotide mimics (unlocked nucleobase        analogs)-3′-Phosphate    -   N_(UNAS)=2′,3′-seco nucleotide mimics (unlocked nucleobase        analogs)-3′-phosphorothioate    -   U_(UNA)=2′,3′-seco-uridine-3′-phosphate    -   U_(UNAS)=2′,3′-seco-uridine-3′-phosphorothioate    -   a_2N=see Table 7    -   a_2Ns=see Table 7    -   pu_2N=see Table 7    -   pu_2Ns=see Table 7    -   Npu=see Table 7    -   Nus=see Table 7    -   N_(LNA)=locked nucleotide    -   Nf_(ANA)=2′-F-Arabino nucleotide    -   NM=2′-methoxyethyl nucleotide    -   AM=2′-methoxyethyladenosine-3′-phosphate    -   AMs=2′-methoxyethyladenosine-3′-phosphorothioate    -   TM=2′-methoxyethylthymidine-3′-phosphate    -   TMs=2′-methoxyethylthymidine-3′-phosphorothioate    -   R=ribitol    -   (invdN)=any inverted deoxyribonucleotide (3′-3′ linked        nucleotide)    -   (invAb)=inverted (3′-3′ linked) abasic deoxyribonucleotide, see        Table 7    -   (invAb)s=inverted (3′-3′ linked) abasic        deoxyribonucleotide-5′-phosphorothioate, see Table 7    -   (invn)=any inverted 2′-OMe nucleotide (3′-3′ linked nucleotide)    -   s=phosphorothioate linkage    -   vpdN=vinyl phosphonate deoxyribonucleotide    -   (5Me-Nf)=5′-Me, 2′-fluoro nucleotide    -   cPrp=cyclopropyl phosphonate, see Table 7    -   epTcPr=see Table 7    -   epTM=see Table 7    -   (Chol-TEG)=see Table 7    -   (TEG-Biotin)=see Table 7    -   (PEG-C3-SS)=see Table 7    -   (Alk-SS-C6)=see Table 7    -   (C6-SS-Alk)=see Table 7    -   (C6-SS-Alk-Me)=see Table 7

The person or ordinary skill in the art would readily understand thatthe terminal nucleotide at the 3″ end of a given oligonucleotidesequence would typically have a hydroxyl (—OH) group at the respective3° position of the given monomer instead of a phosphate moiety ex vivo.Unless expressly indicated otherwise herein, such understandings of theperson of ordinary skill in the art are used when describing the AATRNAi agents and compositions of AAT RNAi agents disclosed herein.

Targeting groups and linking groups include the following, for whichtheir chemical structures are provided below in Table 7: (PAZ), (NAG13),(NAG13)s, (NAG18), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s,(NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29),(NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s,(NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36),(NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), (NAG39)s. Eachsense strand and/or antisense strand can have any targeting groups orlinking groups listed above, as well as other targeting or linkinggroups, conjugated to the 5′ and/or 3′ end of the sequence.

TABLE 4 AAT RNAi Agent Antisense Strand Sequences SEQ Antisense SEQUnderlying Antisense  ID Sequence (Modified) ID Base Sequence Strand ID:NO. (5′ → 3′) NO. (5′ → 3′) AM00516-AS 888dTGfgAfaCfU_(UNA)UfgGfuGfaUfgAfuAfudTsdT 783 TGGAACUUGGUGAUGAUAUTTAM02129-AS 889 dTsGfsgAfaCfU_(UNA)UfgGfuGfaUfgAfuAfudTsdT 783TGGAACUUGGUGAUGAUAUTT AM02130-AS 890dTsGfsgAfaCfU_(UNA)UfgGfuGfaUfgAfuAfuCfgsusg 784 TGGAACUUGGUGAUGAUAUCGUGAM04786-AS 891 aCfU_(UNA)UfgGfuGfaUfgAfuAfudTsdT 785 ACUUGGUGAUGAUAUTTAM05303-AS 892 dTdGdGdAdAdCdTdTdGdGdTdGdAdTdGdAdTdAdTdTsdT 786TGGAACTTGGTGATGATATTT AM05643-AS 893 usUfsusAfaAfcAfUfGfcCfuAfaAfcGfcusu787 UUUAAACAUGCCUAAACGCUU AM05645-AS 894usGfscsAfuUfgCfCfCfaGfgUfaUfuUfcusu 788 UGCAUUGCCCAGGUAUUUCUU AM05647-AS895 usGfsgsAfaCfuUfGfGfuGfaUfgAfuAfuusu 789 UGGAACUUGGUGAUGAUAUUUAM05649-AS 896 usGfsasUfcAfuAfGfGfuucCfaGfuAfausu 790UGAUCAUAGGUUCCAGUAAUU AM05651-AS 897 usAfscsAfgCfcUfUfAfuGfcAfcGfgCfcusu791 UACAGCCUUAUGCACGGCCUU AM05653-AS 898usUfscsGfaUfgGfUfCfaGfcAfcAfgCfcusu 792 UUCGAUGGUCAGCACAGCCUU AM05655-AS899 usCfsasAfaGfgGfUfUfuGfuUfgAfaCfuusu 793 UCAAAGGGUUUGUUGAACUUUAM05657-AS 900 usGfsusUfaAfaCfAfUfgCfcUfaAfaCfgusu 794UGUUAAACAUGCCUAAACGUU AM05659-AS 901 usUfuAfaAfcgugcCfuAfaAfcGfcsUfsg795 UUUAAACGUGCCUAAACGCUG AM05661-AS 902usGfscAfuUfgcccaGfgUfaUfuUfcsAfsg 796 UGCAUUGCCCAGGUAUUUCAG AM05663-AS903 usGfsgAfaCfuugguGfaUfgAfuAfusCfsg 797 UGGAACUUGGUGAUGAUAUCGAM05665-AS 904 usGfsaUfcAfuagguUfcCfaGfuAfasUfsg 798UGAUCAUAGGUUCCAGUAAUG AM05667-AS 905 usAfscAfgCfcuuauGfcAfcGfgCfcsUfsu791 UACAGCCUUAUGCACGGCCUU AM05669-AS 906usUfscGfaUfggucaGfcAfcAfgCfcsUfsu 792 UUCGAUGGUCAGCACAGCCUU AM05671-AS907 usCfsaAfaGfgguuuGfuUfgAfaCfusUfsg 799 UCAAAGGGUUUGUUGAACUUGAM05673-AS 908 usGfsuUfaAfacaugCfcUfaAfaCfgsCfsg 800UGUUAAACAUGCCUAAACGCG AM05677-AS 909 usUfsuAfaAfcgugcCfuAfaAfcGfcsUfsg795 UUUAAACGUGCCUAAACGCUG AM05884-AS 910vpusGfsusUfaAfaCfAfUfgCfcUfaAfaCfgusu 794 UGUUAAACAUGCCUAAACGUUAM05885-AS 911 cPrpusGfsusUfaAfaCfAfUfgCfcUfaAfaCfgusu 794UGUUAAACAUGCCUAAACGUU AM05886-AS 912 usGfsusUfaAfaCfAfUfgCfcUfaAfaCfgcsu801 UGUUAAACAUGCCUAAACGCU AM05887-AS 913usGfsusUfaAfaCfaUfgCfcUfaAfaCfgusu 794 UGUUAAACAUGCCUAAACGUU AM05888-AS914 usGfsusUfaAfaCfaUfgCfcUfaAfaCfgcsu 801 UGUUAAACAUGCCUAAACGCUAM05889-AS 915 usGfsusUfaAfaCfAfUfgCfcUfaAfaCfgCfsu 801UGUUAAACAUGCCUAAACGCU AM05890-AS 916usGfsusUfaAfaCfAfUfgCfcUfaAfaCfgCfuusc 802 UGUUAAACAUGCCUAAACGCUUCAM05891-AS 917 usGfsusUfaAfaCfaUfgCfcUfaAfaCfgCfsu 801UGUUAAACAUGCCUAAACGCU AM05892-AS 918usGfsusUfaAfaCfaUfgCfcUfaAfaCfgCfuusc 802 UGUUAAACAUGCCUAAACGCUUCAM05900-AS 919 cPrpusGfsuUfaAfacaugCfcUfaAfaCfgsCfsg 800UGUUAAACAUGCCUAAACGCG AM05901-AS 920usGfsgsAfaCfU_(UNA)UfGfGfuGfaUfgAfuAfuusu 789 UGGAACUUGGUGAUGAUAUUUAM05954-AS 921 usGfscsUfgUfuggacUfgGfuGfuGfcusu 803UGCUGUUGGACUGGUGUGCUU AM05955-AS 922 usGfscsUfgUfuggacUfgGfuGfuGfccsa804 UGCUGUUGGACUGGUGUGCCA AM05956-AS 923usGfscsUfgUfuggacUfgGfuGfuGfccausu 805 UGCUGUUGGACUGGUGUGCCAUUAM05957-AS 924 usGfscsUfgUfuggacUfgGfuGfuGfccagsc 806UGCUGUUGGACUGGUGUGCCAGC AM05961-AS 925usAfsasGfgCfuUfcUfgAfgUfgGfuAfcusu 807 UAAGGCUUCUGAGUGGUACUU AM05962-AS926 usAfsasGfgCfuUfcUfgAfgUfgGfuAfcasa 808 UAAGGCUUCUGAGUGGUACAAAM05963-AS 927 usAfsasGfgCfuUfcUfgAfgUfgGfuAfcaacsu 809UAAGGCUUCUGAGUGGUACAACU AM05964-AS 928gsAfsasGfgCfuUfcUfgAfgUfgGfuAfcusu 810 GAAGGCUUCUGAGUGGUACUU AM05969-AS929 asAfsgsAfcAfaAfgGfgUfuUfgUfuGfausu 811 AAGACAAAGGGUUUGUUGAUUAM05970-AS 930 asAfsgsAfcAfaAfgGfgUfuUfgUfuGfaasc 812AAGACAAAGGGUUUGUUGAAC AM05973-AS 931 usAfsgsAfcAfaAfgGfgUfuUfgUfuGfaasc813 UAGACAAAGGGUUUGUUGAAC AM05974-AS 932asAfsgsAfcAfaAfgGfgUfuUfgUfuGfaacusu 814 AAGACAAAGGGUUUGUUGAACUUAM05976-AS 933 usAfsgsAfcAfuGfgGfuAfuGfgCfcUfcusu 815UAGACAUGGGUAUGGCCUCUU AM05977-AS 934 usAfsgsAfcAfuGfgGfuAfuGfgCfcUfcusa816 UAGACAUGGGUAUGGCCUCUA AM05979-AS 935usAfsgsAfcAfuGfgGfuAfuGfgCfcUfcuaasa 817 UAGACAUGGGUAUGGCCUCUAAAAM05980-AS 936 usAfsgsAfcAfuGfgGfuAfuGfgCfcUfcuausu 818UAGACAUGGGUAUGGCCUCUAUU AM05982-AS 937usUfsusGfaUfcUfgUfuUfcUfuGfgCfcusu 819 UUUGAUCUGUUUCUUGGCCUU AM05983-AS938 usUfsusGfaUfcUfgUfuUfcUfuGfgCfcusc 820 UUUGAUCUGUUUCUUGGCCUCAM05985-AS 939 usUfsusGfaUfcUfgUfuUfcUfuGfgCfcucusu 821UUUGAUCUGUUUCUUGGCCUCUU AM05987-AS 940 usGfsusUfgGfacuggUfgUfgCfcAfgusu822 UGUUGGACUGGUGUGCCAGUU AM05989-AS 941usGfsusUfgGfacuggUfgUfgCfcAfgcsu 823 UGUUGGACUGGUGUGCCAGCU AM05990-AS942 usGfsusUfgGfacuggUfgUfgCfcAfgcugsg 824 UGUUGGACUGGUGUGCCAGCUGGAM05992-AS 943 usGfsusUfgGfacuggUfgUfgCfcAfgcusg 825UGUUGGACUGGUGUGCCAGCUG AM05994-AS 944 asAfsasGfgGfuUfuGfuUfgAfaCfuUfgusu826 AAAGGGUUUGUUGAACUUGUU AM05996-AS 945asAfsasGfgGfuUfuGfuUfgAfaCfuUfgasc 827 AAAGGGUUUGUUGAACUUGAC AM05998-AS946 usAfsasGfgGfuUfuGfuUfgAfaCfuUfgaccsu 828 UAAGGGUUUGUUGAACUUGACCUAM05999-AS 947 usAfsasGfgGfuUfuGfuUfgAfaCfuUfgasc 829UAAGGGUUUGUUGAACUUGAC AM06124-AS 948 usAfsusUfgGfuGfcUfgUfuGfgAfcUfgusu830 UAUUGGUGCUGUUGGACUGUU AM06125-AS 949usAfsusUfgGfuGfcUfgUfuGfgAfcUfggsu 831 UAUUGGUGCUGUUGGACUGGU AM06126-AS950 usAfsusUfgGfuGfcUfgUfuGfgAfcUfggusu 832 UAUUGGUGCUGUUGGACUGGUUAM06130-AS 951 usUfsgsUfuGfgacugGfuGfuGfcCfasg 833 UUGUUGGACUGGUGUGCCAGAM06131-AS 952 usUfsgsUfuGfgacugGfuGfuGfcCfagcsu 834UUGUUGGACUGGUGUGCCAGCU AM06133-AS 953 usAfsusAfgAfcAfuGfgGfuAfuGfgCfcusc835 UAUAGACAUGGGUAUGGCCUC AM06134-AS 954usAfsusAfgAfcauggGfuAfuGfgCfcusc 835 UAUAGACAUGGGUAUGGCCUC AM06137-AS955 usCfsasAfaGfgGfuUfuGfuUfgAfaCfuugasc 836 UCAAAGGGUUUGUUGAACUUGACAM06140-AS 956 usUfsasUfuGfgugcuGfuUfgGfaCfugsg 837UUAUUGGUGCUGUUGGACUGG AM06227-AS 957 usGfsusUfaAfaCfaUfgCfcUfaAfaCfgsc838 UGUUAAACAUGCCUAAACGC AM06228-AS 958usGfsusUfaAfaCfaUfgCfcUfaAfaCfgcusu 839 UGUUAAACAUGCCUAAACGCUUAM06234-AS 959 usGfsuUfaAfaCfaUfgCfcUfaAfaCfgsCfsg 800UGUUAAACAUGCCUAAACGCG AM06235-AS 960 usGfsuUfaAfacaugCfcUfaAfaCfgCfsu801 UGUUAAACAUGCCUAAACGCU AM06237-AS 961usGfsuUfaAfaCfAfUfgCfcUfaAfaCfgsCfsg 800 UGUUAAACAUGCCUAAACGCGAM06238-AS 962 NpusGfsusUfaAfaCfaUfgCfcUfaAfaCfgusu 794UGUUAAACAUGCCUAAACGUU AM06261-AS 963 NusGfsusUfaAfaCfaUfgCfcUfaAfaCfgusu794 UGUUAAACAUGCCUAAACGUU

TABLE 5 AAT RNAi Agent Sense Strand Sequences Sense SEQ Sense SEQUnderlying Strand ID Sequence (Modified) ID Base Sequence ID: NO.(5′ → 3′) NO. (5′ → 3′) AM01887-SS  964(Chol-TEG)uAuAfuAfuCfaUfcAfcCfaAfgUfuCfcAf 845 UAUAUAUCAUCACCAAGUUCCAT(invdT)(TEG-Biotin) AM01888-SS  965(Chol-TEG)uAuAfuAfuCfaUfcAfcCfaAfgUfuCfcAf 845 UAUAUAUCAUCACCAAGUUCCAT(invdT)(PEG-C3-SS) AM01855-SS  966(Alk-SS-C6)AfuAfuCfaUfcAfcCfaAfgUfuCfcAf(invdT) 846 AUAUCAUCACCAAGUUCCATAM02132-SS  967 CfsgsAfuAfuCfaUfcAfcCfaAfgUfuCfcAf(C6-SS-Alk) 847CGAUAUCAUCACCAAGUUCCA AM02390-SS  968CfsgsAfuAfuCfaUfcAfcCfaAfgUfuCfcAf(C6-SS-Alk-Me) 847CGAUAUCAUCACCAAGUUCCA AM04785-SS  969 uAfuCfaUfcAfcCfaAfgUfuCfcAf(invdT)848 UAUCAUCACCAAGUUCCAT AM05304-SS  970(Chol-TEG)dTdAdTdAdTdAdTdCdAdTdCdAdCdCdAdAdGdTdTdCd 849TATATATCATCACCAAGTTCCAT CsdA(invdT)(TEG-Biotin) AM05599-SS  971(Chol-TEG)dTdAdTdAdTdAdTdCdAdTdCdAdCdCdAdAdGdTdTdCd 849TATATATCATCACCAAGTTCCAT CsdA(invdT) AM05642-SS  972(NAG25)(invAb)GfcGfuUfuAfGfGfcAfuGfuUfuAfaausu(invAb) 850GCGUUUAGGCAUGUUUAAAUU AM05644-SS  973(NAG25)(invAb)GfaAfaUfaCfCfUfgGfgCfaAfuGfcausu(invAb) 851GAAAUACCUGGGCAAUGCAUU AM05646-SS  974(NAG25)(invAb)AfuAfuCfaUfCfAfcCfaAfgUfuCfcausu(invAb) 852AUAUCAUCACCAAGUUCCAUU AM05648-SS  975(NAG25)(invAb)UfuAfcUfgGfAfAfcCfuAfuGfaUfcausu(invAb) 853UUACUGGAACCUAUGAUCAUU AM05650-SS  976(NAG25)(invAb)GfgCfcGfuGfCfAfuAfaGfgCfuGfuausu(invAb) 854GGCCGUGCAUAAGGCUGUAUU AM05652-SS  977(NAG25)(invAb)GfgCfuGfuGfCfUfgAfcCfaUfcGfaausu(invAb) 855GGCUGUGCUGACCAUCGAAUU AM05654-SS  978(NAG25)(invAb)AfgUfuCfaAfCfAfaAfcCfcUfuUfgausu(invAb) 856AGUUCAACAAACCCUUUGAUU AM05656-SS  979(NAG25)(invAb)CfgUfuUfaGfGfCfaUfgUfuUfaAfcausu(invAb) 857CGUUUAGGCAUGUUUAACAUU AM05658-SS  980(NAG25)scsagcguuuAfGfGfcauguuuaasa(invAb) 858 CAGCGUUUAGGCAUGUUUAAAAM05660-SS  981 (NAG25)scsugaaauaCfCfUfgggcaaugcsa(invAb) 859CUGAAAUACCUGGGCAAUGCA AM05662-SS  982(NAG25)scsgauaucaUfCfAfccaaguuccsa(invAb) 847 CGAUAUCAUCACCAAGUUCCAAM05664-SS  983 (NAG25)scsauuacugGfAfAfccuaugaucsa(invAb) 860CAUUACUGGAACCUAUGAUCA AM05666-SS  984(NAG25)sasaggccguGfCfAfuaaggcugusa(invAb) 861 AAGGCCGUGCAUAAGGCUGUAAM05668-SS  985 (NAG25)sasaggcuguGfCfUfgaccaucgasa(invAb) 862AAGGCUGUGCUGACCAUCGAA AM05670-SS  986(NAG25)scsaaguucaAfCfAfaacccuuugsa(invAb) 863 CAAGUUCAACAAACCCUUUGAAM05672-SS  987 (NAG25)scsgcguuuaGfGfCfauguuuaacsa(invAb) 864CGCGUUUAGGCAUGUUUAACA AM05658-SS  988(NAG25)scsagcguuuAfGfGfcauguuuaasa(invAb) 858 CAGCGUUUAGGCAUGUUUAAAAM05893-SS  989 (NAG25)s(invAb)scguuuaGfGfCfauguuuaacausu(invAb) 857CGUUUAGGCAUGUUUAACAUU AM05894-SS  990(NAG25)s(invAb)sCfgUfuUfaGfGfCfaUfgUfuUfaAfcas(invAb) 429CGUUUAGGCAUGUUUAACA AM05895-SS  991(NAG25)s(invAb)scguuuaGfGfCfauguuuaacas(invAb) 429 CGUUUAGGCAUGUUUAACAAM05896-SS  992 (NAG25)s(invAb)saaCfgUfuUfaGfGfCfaUfgUfuUfaAfcas 865AACGUUUAGGCAUGUUUAACA (invAb) AM05897-SS  993(NAG25)s(invAb)sagCfgUfuUfaGfGfCfaUfgUfuUfaAfcas 866AGCGUUUAGGCAUGUUUAACA (invAb) AM05898-SS  994(NAG25)s(invAb)saacguuuaGfGfCfauguuuaacas(invAb) 865AACGUUUAGGCAUGUUUAACA AM05899-SS  995(NAG25)s(invAb)sagcguuuaGfGfCfauguuuaacas(invAb) 866AGCGUUUAGGCAUGUUUAACA AM05958-SS  996(NAG37)s(invAb)sgcacacCfAfGfuccaacagcas(invAb) 454 GCACACCAGUCCAACAGCAAM05959-SS  997 (NAG37)s(invAb)suggcacacCfAfGfuccaacagcas(invAb) 867UGGCACACCAGUCCAACAGCA AM05960-SS  998(NAG37)s(invAb)saagcacacCfAfGfuccaacagcas(invAb) 868AAGCACACCAGUCCAACAGCA AM05965-SS  999(NAG37)s(invAb)sguaccaCfUfCfagaagccuuas(invAb) 519 GUACCACUCAGAAGCCUUAAM05966-SS 1000 (NAG37)s(invAb)suuguaccaCfUfCfagaagccuuas(invAb) 869UUGUACCACUCAGAAGCCUUA AM05967-SS 1001(NAG37)s(invAb)sguaccaCfUfCfagaagccuucs(invAb) 518 GUACCACUCAGAAGCCUUCAM05968-SS 1002 (NAG37)s(invAb)sucaacaAfAfCfccuuugucuus(invAb) 738UCAACAAACCCUUUGUCUU AM05971-SS 1003(NAG37)s(invAb)sguucaacaAfAfCfccuuugucuus(invAb) 870GUUCAACAAACCCUUUGUCUU AM05972-SS 1004(NAG37)s(invAb)sguucaacaAfAfCfccuuugucuas(invAb) 871GUUCAACAAACCCUUUGUCUA AM05975-SS 1005(NAG37)s(invAb)sgaggccAfUfAfcccaugucuas(invAb) 707 GAGGCCAUACCCAUGUCUAAM05978-SS 1006 (NAG37)s(invAb)suagaggccAfUfAfcccaugucuas(invAb) 872UAGAGGCCAUACCCAUGUCUA AM05981-SS 1007(NAG37)s(invAb)sggccaaGfAfAfacagaucaaas(invAb) 537 GGCCAAGAAACAGAUCAAAAM05984-SS 1008 (NAG37)s(invAb)sgaggccaaGfAfAfacagaucaaas(invAb) 873GAGGCCAAGAAACAGAUCAAA AM05986-SS 1009(NAG37)s(invAb)scuggcaCfAfCfcaguccaacas(invAb) 445 CUGGCACACCAGUCCAACAAM05988-SS 1010 (NAG37)s(invAb)sagcuggcaCfAfCfcaguccaacas(invAb) 874AGCUGGCACACCAGUCCAACA AM05991-SS 1011(NAG37)s(invAb)sgcuggcaCfAfCfcaguccaacas(invAb) 875 GCUGGCACACCAGUCCAACAAM05993-SS 1012 (NAG37)s(invAb)scaaguuCfAfAfcaaacccuuus(invAb) 725CAAGUUCAACAAACCCUUU AM05995-SS 1013(NAG37)s(invAb)sgucaaguuCfAfAfcaaacccuuus(invAb) 876GUCAAGUUCAACAAACCCUUU AM05997-SS 1014(NAG37)s(invAb)sgucaaguuCfAfAfcaaacccuuas(invAb) 877GUCAAGUUCAACAAACCCUUA AM06127-SS 1015(NAG37)s(invAb)scaguccAfAfCfagcaccaauas(invAb) 458 CAGUCCAACAGCACCAAUAAM06128-SS 1016 (NAG37)s(invAb)saccaguccAfAfCfagcaccaauas(invAb) 878ACCAGUCCAACAGCACCAAUA AM06129-SS 1017(NAG37)s(invAb)sccaguccAfAfCfagcaccaauas(invAb) 879 CCAGUCCAACAGCACCAAUAAM06132-SS 1018 (NAG37)s(invAb)scuggcacAfCfCfaguccaacaas(invAb) 880CUGGCACACCAGUCCAACAA AM06135-SS 1019(NAG37)s(invAb)sggccauAfCfCfcaugucuauas(invAb) 712 GGCCAUACCCAUGUCUAUAAM06136-SS 1020 (NAG37)s(invAb)sgaggccauAfCfCfcaugucuauas(invAb) 881GAGGCCAUACCCAUGUCUAUA AM06138-SS 1021(NAG37)s(invAb)sgucaaguucaAfCfAfaacccuuugas(invAb) 882GUCAAGUUCAACAAACCCUUUGA AM06139-SS 1022(NAG37)s(invAb)scaaguucaAfCfAfaacccuuugas(invAb) 863CAAGUUCAACAAACCCUUUGA AM06141-SS 1023(NAG37)s(invAb)sccaguccaAfCfAfgcaccaauaas(invAb) 883CCAGUCCAACAGCACCAAUAA AM06195-SS 1024(NAG37)s(invAb)scgcguuuaGfGfCfauguuuaacas(invAb) 864CGCGUUUAGGCAUGUUUAACA AM06223-SS 1025(NAG37)s(invAb)scguuuaGfGfCfauguuuaacas(invAb) 429 CGUUUAGGCAUGUUUAACAAM06224-SS 1026 (NAG37)s(invAb)scsgcguuuaGfGfCfauguuuaacsa(invAb) 864CGCGUUUAGGCAUGUUUAACA AM06225-SS 1027(NAG37)s(invAb)sagCfgUfuUfaGfGfCfaUfgUfuUfaAfcas 866AGCGUUUAGGCAUGUUUAACA (invAb) AM06226-SS 1028(NAG37)s(invAb)scguuuaGfGfCfauguuuaacausu(invAb) 857CGUUUAGGCAUGUUUAACAUU AM06229-SS 1029(NAG37)s(invAb)sgcguuuaGfGfCfauguuuaacas(invAb) 884 GCGUUUAGGCAUGUUUAACAAM06230-SS 1030 (NAG37)s(invAb)sgcguuuaGfGfCfauguuuaacausu(invAb) 885GCGUUUAGGCAUGUUUAACAUU AM06231-SS 1031(NAG37)s(invAb)scgcguuuaGfGfCfauguuuaacsausu(invAb) 886CGCGUUUAGGCAUGUUUAACAUU AM06232-SS 1032(NAG37)s(invAb)sagCfgUfuUfaGfGfCfaUfgUfuUfaAfcausu 887AGCGUUUAGGCAUGUUUAACAUU (invAb) AM06236-SS 1033(NAG37)s(invAb)sagcguuuaGfGfCfauguuuaacas(invAb) 866AGCGUUUAGGCAUGUUUAACA AM06239-SS 1034(NAG37)s(invAb)scgcguuuaGfGfCfauguuuaacsas(invAb) 864CGCGUUUAGGCAUGUUUAACA

The AAT RNAi agents described herein are formed by annealing anantisense strand with a sense strand. A sense strand containing asequence listed in Table 2, Table 3, or Table 5 can be hybridized to anyantisense strand containing a sequence listed in Table 2, Table 3, orTable 4, provided the two sequences have a region of at least 85%complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotidesequence.

In some embodiments, the antisense strand of an AAT RNAi agent disclosedherein differs by 0, 1, 2, or 3 nucleotides from any of the antisensestrand sequences in Table 4. In some embodiments, the sense strand of anAAT RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotidesfrom any of the sense strand sequences in Table 5.

In some embodiments, an AAT RNAi agent antisense strand comprises anucleotide sequence of any of the sequences in Table 2, Table 3, orTable 4. In some embodiments, an AAT RNAi agent antisense strandcomprises the sequence of nucleotides (from 5′ end→3′ end) 1-17, 2-17,1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23,1-24, or 2-24, of any of the sequences in Table 2, Table 3, or Table 4.In certain embodiments, an AAT RNAi agent antisense strand comprises orconsists of a modified sequence of any one of the modified sequences inTable 4.

In some embodiments, an AAT RNAi agent sense strand comprises thenucleotide sequence of any of the sequences in Table 2, Table 3, orTable 5. In some embodiments, an AAT RNAi agent sense strand comprisesthe sequence of nucleotides (from 5′ end→3′ end) 1-17, 2-17, 3-17, 4-17,1-18, 2-18, 3-18, 4-18, 1-19, 2-19, 3-19, 4-19, 1-20, 2-20, 3-20, 4-20,1-21, 2-21, 3-21, 4-21, 1-22, 2-22, 3-22, 4-22, 1-23, 2-23, 3-23, 4-23,1-24, 2-24, 3-24, or 4-24 of any of the sequences in Table 2, Table 3,or Table 5. In certain embodiments, an AAT RNAi agent sense strandcomprises or consists of a modified sequence of any one of the modifiedsequences in Table 5.

For the AAT RNAi agents disclosed herein, the nucleotide at position 1of the antisense strand (from 5′ end→3′ end) can be perfectlycomplementary to an AAT gene, or can be non-complementary to an AATgene. In some embodiments, the nucleotide at position 1 of the antisensestrand (from 5′ end→3′ end) is a U, A, or dT (or a modified version ofU, A or dT). In some embodiments, the nucleotide at position 1 of theantisense strand (from 5′ end→3′ end) forms an A:U or U:A base pair withthe sense strand.

In some embodiments, an AAT RNAi agent antisense strand comprises thesequence of nucleotides (from 5′ end→3′ end) 2-18 or 2-19 of any of theantisense strand sequences in Table 2, Table 3, or Table 4. In someembodiments, an AAT RNAi sense strand comprises the sequence ofnucleotides (from 5′ end→3′ end) 1-17 or 1-18 of any of the sense strandsequences in Table 2, Table 3, or Table 5.

In some embodiments, an AAT RNAi agent includes (i) an antisense strandcomprising the sequence of nucleotides (from 5′ end→3′ end) 2-18 or 2-19of any of the antisense strand sequences in Table 2, Table 3, or Table4, and (ii) a sense strand comprising the sequence of nucleotides (from5′ end→3′ end) 1-17 or 1-18 of any of the sense strand sequences inTable 2, Table 3, or Table 5.

A sense strand containing a sequence listed in Table 2, Table 3, orTable 5 can be hybridized to any antisense strand containing a sequencelisted in Table 2, Table 3, or Table 5, provided the two sequences havea region of at least 85% complementarity over a contiguous 16, 17, 18,19, 20, or 21 nucleotide sequence. In some embodiments, the AAT RNAiagent has a sense strand consisting of the modified sequence of any ofthe modified sequences in Table 5, and an antisense strand consisting ofthe modified sequence of any of the modified sequences in Table 4.Representative sequence pairings are exemplified by the Duplex ID Nos.shown in Table 6.

In some embodiments, an AAT RNAi agent comprises any of the duplexesrepresented by any of the Duplex ID Nos. presented herein. In someembodiments, an AAT RNAi agent consists of any of the duplexesrepresented by any of the Duplex ID Nos. presented herein. In someembodiments, an AAT RNAi agent comprises the sense strand and antisensestrand nucleotide sequences of any of the duplexes represented by any ofthe Duplex ID Nos. presented herein. In some embodiments, an AAT RNAiagent includes the sense strand and antisense strand nucleotidesequences of any of the duplexes represented by any of the Duplex IDNos. presented herein and a targeting group and/or linking group,wherein the targeting group and/or linking group is covalently linked(i.e., conjugated) to the sense strand or the antisense strand. In someembodiments, an AAT RNAi agent includes the sense strand and antisensestrand modified nucleotide sequences of any of the duplexes representedby any of the Duplex ID Nos. presented herein. In some embodiments, anAAT RNAi agent comprises the sense strand and antisense strand modifiednucleotide sequences of any of the duplexes represented by any of theDuplex ID Nos. presented herein and a targeting group and/or linkinggroup, wherein the targeting group and/or linking group is covalentlylinked to the sense strand or the antisense strand.

In some embodiments, an AAT RNAi agent comprises an antisense strand anda sense strand having the nucleotide sequences of any of the antisensestrand/sense strand duplexes of Table 2, Table 3, or Table 6, andcomprises an asialoglycoprotein receptor ligand targeting group.

In some embodiments, an AAT RNAi agent comprises an antisense strand anda sense strand having the nucleotide sequences of any of the antisensestrand/sense strand duplexes of Table 2 or Table 5, and furthercomprises a targeting group selected from the group consisting of (PAZ),(NAG13), (NAG13)s, (NAG18), (NAG18)s, (NAG24), (NAG24)s, (NAG25),(NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s,(NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32),(NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s,(NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39),(NAG39)s. In some embodiments, the targeting group is (NAG25) or(NAG25)s as defined in Table 7. In other embodiments, the targetinggroup is (NAG37) or (NAG37)s as defined in Table 7.

In some embodiments, an AAT RNAi agent comprises an antisense strand anda sense strand having the modified nucleotide sequence of any of theantisense strand and/or sense strand nucleotide sequences of any of theduplexes of Table 6.

In some embodiments, an AAT RNAi agent comprises an antisense strand anda sense strand having a modified nucleotide sequence of any of theantisense strand and/or sense strand nucleotide sequences of any of theduplexes of Table 6, and comprises an asialoglycoprotein receptor ligandtargeting group.

In some embodiments, an AAT RNAi agent comprises the duplex structure ofany of the duplexes in Table 6.

In some embodiments, an AAT RNAi agent consists of the duplex structureof any of the duplexes in Table 6.

TABLE 6 AAT RNAi Agents Identified by Duplex ID No. with CorrespondingSense and Antisense Strands Duplex Antisense Sense ID Strand ID StrandID AD01131 AM00516-AS AM01887-SS AD01132 AM00516-AS AM01888-SS AD01174AM00516-AS AM01855-SS AD01286 AM02129-AS AM01855-SS AD01287 AM02130-ASAM02132-SS AD01442 AM02130-AS AM02390-SS AD03752 AM04786-AS AM04785-SSAD04156 AM05303-AS AM05304-SS AD04406 AM05303-AS AM05599-SS AD04444AM05643-AS AM05642-SS AD04445 AM05645-AS AM05644-SS AD04446 AM05647-ASAM05646-SS AD04447 AM05649-AS AM05648-SS AD04448 AM05651-AS AM05650-SSAD04449 AM05653-AS AM05652-SS AD04450 AM05655-AS AM05654-SS AD04451AM05657-AS AM05656-SS AD04452 AM05659-AS AM05658-SS AD04453 AM05661-ASAM05660-SS AD04454 AM05663-AS AM05662-SS AD04455 AM05665-AS AM05664-SSAD04456 AM05667-AS AM05666-SS AD04457 AM05669-AS AM05668-SS AD04458AM05671-AS AM05670-SS AD04459 AM05673-AS AM05672-SS AD04464 AM05677-ASAM05658-SS AD04601 AM05884-AS AM05656-SS AD04602 AM05885-AS AM05656-SSAD04603 AM05886-AS AM05656-SS AD04604 AM05887-AS AM05893-SS AD04605AM05888-AS AM05893-SS AD04606 AM05657-AS AM05894-SS AD04607 AM05886-ASAM05894-SS AD04608 AM05887-AS AM05895-SS AD04609 AM05888-AS AM05895-SSAD04610 AM05657-AS AM05896-SS AD04611 AM05889-AS AM05897-SS AD04612AM05890-AS AM05897-SS AD04613 AM05887-AS AM05898-SS AD04614 AM05891-ASAM05899-SS AD04615 AM05892-AS AM05899-SS AD04616 AM05900-AS AM05672-SSAD04617 AM05901-AS AM05646-SS AD04652 AM05954-AS AM05958-SS AD04653AM05955-AS AM05958-SS AD04654 AM05955-AS AM05959-SS AD04655 AM05954-ASAM05960-SS AD04656 AM05956-AS AM05959-SS AD04657 AM05957-AS AM05959-SSAD04658 AM05961-AS AM05965-SS AD04659 AM05962-AS AM05965-SS AD04660AM05962-AS AM05966-SS AD04661 AM05963-AS AM05966-SS AD04662 AM05964-ASAM05967-SS AD04663 AM05969-AS AM05968-SS AD04664 AM05970-AS AM05968-SSAD04665 AM05970-AS AM05971-SS AD04666 AM05973-AS AM05972-SS AD04667AM05974-AS AM05971-SS AD04668 AM05976-AS AM05975-SS AD04669 AM05977-ASAM05975-SS AD04670 AM05977-AS AM05978-SS AD04671 AM05979-AS AM05978-SSAD04672 AM05980-AS AM05978-SS AD04673 AM05982-AS AM05981-SS AD04674AM05983-AS AM05981-SS AD04675 AM05983-AS AM05984-SS AD04676 AM05985-ASAM05984-SS AD04677 AM05987-AS AM05986-SS AD04678 AM05989-AS AM05988-SSAD04679 AM05990-AS AM05988-SS AD04680 AM05992-AS AM05991-SS AD04681AM05994-AS AM05993-SS AD04682 AM05996-AS AM05995-SS AD04683 AM05998-ASAM05997-SS AD04684 AM05999-AS AM05997-SS AD04761 AM06124-AS AM06127-SSAD04762 AM06125-AS AM06128-SS AD04763 AM06126-AS AM06129-SS AD04764AM06130-AS AM06132-SS AD04765 AM06131-AS AM06132-SS AD04766 AM06133-ASAM06135-SS AD04767 AM06134-AS AM06136-SS AD04768 AM06137-AS AM06138-SSAD04769 AM06137-AS AM06139-SS AD04770 AM06140-AS AM06141-SS AD04805AM05673-AS AM06195-SS AD04824 AM05887-AS AM06223-SS AD04825 AM05900-ASAM06224-SS AD04826 AM05889-AS AM06225-SS AD04827 AM05888-AS AM06223-SSAD04828 AM05887-AS AM06226-SS AD04829 AM06227-AS AM06229-SS AD04830AM06228-AS AM06229-SS AD04831 AM06228-AS AM06230-SS AD04832 AM05673-ASAM06231-SS AD04833 AM05889-AS AM06232-SS AD04834 AM06227-AS AM06230-SSAD04836 AM06234-AS AM06195-SS AD04837 AM06235-AS AM06236-SS AD04838AM06237-AS AM06239-SS AD04839 AM05673-AS AM06239-SS AD04840 AM06238-ASAM06223-SS AD04857 AM06261-AS AM06223-SS

In some embodiments, an AAT RNAi agent is prepared or provided as asalt, mixed salt, or a free-acid. The RNAi agents described herein, upondelivery to a cell expressing an AAT gene, inhibit or knockdownexpression of one or more AAT genes in vivo.

Targeting Groups, Linking Groups, and Delivery Vehicles

In some embodiments, an AAT RNAi agent is conjugated to one or morenon-nucleotide groups including, but not limited to, a targeting group,linking group, delivery polymer, or a delivery vehicle. Thenon-nucleotide group can enhance targeting, delivery or attachment ofthe RNAi agent. Examples of targeting groups and linking groups areprovided in Table 7. The non-nucleotide group can be covalently linkedto the 3′ and/or 5′ end of either the sense strand and/or the antisensestrand. In some embodiments, an AAT RNAi agent contains a non-nucleotidegroup linked to the 3′ and/or 5′ end of the sense strand. In someembodiments, a non-nucleotide group is linked to the 5′ end of an AATRNAi agent sense strand. A non-nucleotide group can be linked directlyor indirectly to the RNAi agent via a linker/linking group. In someembodiments, a non-nucleotide group is linked to the RNAi agent via alabile, cleavable, or reversible bond or linker.

In some embodiments, a non-nucleotide group enhances the pharmacokineticor biodistribution properties of an RNAi agent or conjugate to which itis attached to improve cell- or tissue-specific distribution andcell-specific uptake of the RNAi agent or conjugate. In someembodiments, a non-nucleotide group enhances endocytosis of the RNAiagent.

Targeting groups or targeting moieties can enhance the pharmacokineticor biodistribution properties of a conjugate or RNAi agent to which theyare attached to improve cell-specific distribution and cell-specificuptake of the conjugate or RNAi agent. A targeting group can bemonovalent, divalent, trivalent, tetravalent, or have higher valency forthe target to which it is directed. Representative targeting groupsinclude, without limitation, compounds with affinity to cell surfacemolecules, cell receptor ligands, haptens, antibodies, monoclonalantibodies, antibody fragments, and antibody mimics with affinity tocell surface molecules. In some embodiments, a targeting group is linkedto an RNAi agent using a linker, such as a PEG linker or one, two, orthree abasic and/or ribitol (abasic ribose) residues, which in someinstances can serve as linkers. In some embodiments, a targeting groupcomprises a galactose derivative cluster.

The AAT RNAi agents described herein can be synthesized having areactive group, such as an amine group, at the 5′-terminus. The reactivegroup can be used to subsequently attach a targeting group using methodstypical in the art.

In some embodiments, a targeting group comprises an asialoglycoproteinreceptor ligand. In some embodiments, an asialoglycoprotein receptorligand includes or consists of one or more galactose derivatives. Asused herein, the term galactose derivative includes both galactose andderivatives of galactose having affinity for the asialoglycoproteinreceptor that is equal to or greater than that of galactose. Galactosederivatives include, but are not limited to: galactose, galactosamine,N-formylgalactosamine, N-acetyl-galactosamine,N-propionyl-galactosamine, N-n-butanoyl-galactosamine, andN-iso-butanoylgalactos-amine (see for example: S.T. Iobst and K.Drickamer, J. B. C., 1996, 271, 6686). Galactose derivatives, andclusters of galactose derivatives, that are useful for in vivo targetingof oligonucleotides and other molecules to the liver are known in theart (see, for example, Baenziger and Fiete, 1980, Cell, 22, 611-620;Connolly et al., 1982, J. Biol. Chem., 257, 939-945).

Galactose derivatives have been used to target molecules to hepatocytesin vivo through their binding to the asialoglycoprotein receptorexpressed on the surface of hepatocytes. Binding of asialoglycoproteinreceptor ligands to the asialoglycoprotein receptor(s) facilitatescell-specific targeting to hepatocytes and endocytosis of the moleculeinto hepatocytes. Asialoglycoprotein receptor ligands can be monomeric(e.g., having a single galactose derivative) or multimeric (e.g., havingmultiple galactose derivatives). The galactose derivative or galactosederivative cluster can be attached to the 3′ or 5′ end of the sense orantisense strand of the RNAi agent using methods known in the art. Thepreparation of targeting groups, such as galactose derivative clusters,is described in, for example, U.S. patent application Ser. No.15/452,324 and U.S. Patent Publication No. US 2017/0253875, the contentsof both of which are incorporated by reference herein in their entirety.

As used herein, a galactose derivative cluster comprises a moleculehaving two to four terminal galactose derivatives. A terminal galactosederivative is attached to a molecule through its C-1 carbon. In someembodiments, the galactose derivative cluster is a galactose derivativetrimer (also referred to as tri-antennary galactose derivative ortri-valent galactose derivative). In some embodiments, the galactosederivative cluster comprises N-acetyl-galactosamines. In someembodiments, the galactose derivative cluster comprises threeN-acetyl-galactosamines. In some embodiments, the galactose derivativecluster is a galactose derivative tetramer (also referred to astetra-antennary galactose derivative or tetra-valent galactosederivative). In some embodiments, the galactose derivative clustercomprises four N-acetyl-galactosamines.

As used herein, a galactose derivative trimer contains three galactosederivatives, each linked to a central branch point. As used herein, agalactose derivative tetramer contains four galactose derivatives, eachlinked to a central branch point. The galactose derivatives can beattached to the central branch point through the C-1 carbons of thesaccharides. In some embodiments, the galactose derivatives are linkedto the branch point via linkers or spacers. In some embodiments, thelinker or spacer is a flexible hydrophilic spacer, such as a PEG group(see, for example, U.S. Pat. No. 5,885,968; Biessen et al. J. Med. Chem.1995 Vol. 39 p. 1538-1546). In some embodiments, the PEG spacer is aPEGS spacer. The branch point can be any small molecule which permitsattachment of three galactose derivatives and further permits attachmentof the branch point to the RNAi agent. An example of branch point groupis a di-lysine or di-glutamate. Attachment of the branch point to theRNAi agent can occur through a linker or spacer. In some embodiments,the linker or spacer comprises a flexible hydrophilic spacer, such as,but not limited to, a PEG spacer. In some embodiments, the linkercomprises a rigid linker, such as a cyclic group. In some embodiments, agalactose derivative comprises or consists of N-acetyl-galactosamine. Insome embodiments, the galactose derivative cluster is comprised of agalactose derivative tetramer, which can be, for example, anN-acetyl-galactosamine tetramer.

Embodiments of the present disclosure include pharmaceuticalcompositions for delivering an AAT RNAi agent to a liver cell in vivo.Such pharmaceutical compositions can include, for example, an AAT RNAiagent conjugated to a galactose derivative cluster. In some embodiments,the galactose derivative cluster is comprised of a galactose derivativetrimer, which can be, for example, an N-acetyl-galactosamine trimer, orgalactose derivative tetramer, which can be, for example, anN-acetyl-galactosamine tetramer.

Targeting groups include, but are not limited to, (PAZ), (NAG13),(NAG13)s, (NAG18), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s,(NAG26), (NAG26)s, (NAG27) (NAG27)s, (NAG28) (NAG28)s, (NAG29) (NAG29)s,(NAG30) (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33),(NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s,(NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), and (NAG39)s as definedin Table 7. Other targeting groups, including galactose clustertargeting ligands, are known in the art.

In some embodiments, a linking group is conjugated to the RNAi agent.The linking group facilitates covalent linkage of the agent to atargeting group or delivery polymer or delivery vehicle. The linkinggroup can be linked to the 3′ or the 5′ end of the RNAi agent sensestrand or antisense strand. In some embodiments, the linking group islinked to the RNAi agent sense strand. In some embodiments, the linkinggroup is conjugated to the 5′ or 3′ end of an RNAi agent sense strand.In some embodiments, a linking group is conjugated to the 5′ end of anRNAi agent sense strand. Examples of linking groups, can include, butare not limited to: reactive groups such a primary amines and alkynes,alkyl groups, abasic nucleotides, ribitol (abasic ribose), and/or PEGgroups.

A linker or linking group is a connection between two atoms that linksone chemical group (such as an RNAi agent) or segment of interest toanother chemical group (such as a targeting group or delivery polymer)or segment of interest via one or more covalent bonds. A labile linkagecontains a labile bond. A linkage may optionally include a spacer thatincreases the distance between the two joined atoms. A spacer canfurther add flexibility and/or length to the linkage. Spacers caninclude, but are not be limited to, alkyl groups, alkenyl groups,alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, andaralkynyl groups; each of which can contain one or more heteroatoms,heterocycles, amino acids, nucleotides, and saccharides. Spacer groupsare well known in the art and the preceding list is not meant to limitthe scope of the description.

Any of the AAT RNAi agent nucleotide sequences listed in Tables 2, 3, 4,or 5, whether modified or unmodified, may contain 3′ or 5′ targetinggroups or linking groups. Any of the AAT RNAi agent sequences listed inTables 4 or 5 which contain a 3′ or 5′ targeting group or linking group,may alternatively contain no 3′ or 5′ targeting group or linking group,or may contain a different 3′ or 5′ targeting group or linking groupincluding, but not limited to, those depicted in Table 7. Any of the AATRNAi agent duplexes listed in Table 2, Table 3, or Table 6, whethermodified or unmodified, may further comprise a targeting group orlinking group, including, but not limited to, those depicted in Table 7,and the targeting group or linking group may be attached to the 3′ or 5′terminus of either the sense strand or the antisense strand of the AATRNAi agent duplex.

Examples of targeting groups and linking groups are provided in Table 7.Table 5 provides several embodiments of AAT RNAi agent sense strandshaving a targeting group or linking group linked to the 5′ or 3′ end.

TABLE 7 Structures Representing Various Modified Nucleotides, TargetingGroups, and Linking Groups

When positioned internally on oligonucleotide:

When positioned internally on oligonucleotide:

When positioned at the 3′ terminal end of oligonucleotide

In each of the above structures in Table 7, NAG comprises anN-acetyl-galactosamine or another asialoglycoprotein receptor ligand, aswould be understood by a person of ordinary skill in the art to beattached in view of the structures above and description providedherein. For example, in some embodiments, NAG in the structures providedin Table 7 is represented by the following structure:

Each (NAGx) can be attached to an AAT RNAi agent via a phosphate group(as in (NAG25), (NAG30), and (NAG31)), or a phosphorothioate group, (asis (NAG25)s, (NAG29)s, (NAG30)s, (NAG31)s, or (NAG37)s), or anotherlinking group.

Other linking groups known in the art may be used.

In some embodiments, a delivery vehicle can be used to deliver an RNAiagent to a cell or tissue. A delivery vehicle is a compound thatimproves delivery of the RNAi agent to a cell or tissue. A deliveryvehicle can include, or consist of, but is not limited to: a polymer,such as an amphipathic polymer, a membrane active polymer, a peptide, amelittin peptide, a melittin-like peptide (MLP), a lipid, a reversiblymodified polymer or peptide, or a reversibly modified membrane activepolyamine. In some embodiments, the RNAi agents can be combined withlipids, nanoparticles, polymers, liposomes, micelles, DPCs or otherdelivery systems available in the art. The RNAi agents can also bechemically conjugated to targeting groups, lipids (including, but notlimited to cholesterol and cholesteryl derivatives), nanoparticles,polymers, liposomes, micelles, DPCs (see, for example WO 2000/053722, WO2008/0022309, WO 2011/104169, and WO 2012/083185, WO 2013/032829, WO2013/158141, each of which is incorporated herein by reference), orother delivery systems available in the art.

Pharmaceutical Compositions and Formulations

The AAT RNAi agents disclosed herein can be prepared as pharmaceuticalcompositions or formulations. In some embodiments, pharmaceuticalcompositions include at least one AAT RNAi agent. These pharmaceuticalcompositions are particularly useful in the inhibition of the expressionof the target mRNA in a target cell, a group of cells, a tissue, or anorganism. The pharmaceutical compositions can be used to treat a subjecthaving a disease or disorder that would benefit from reduction in thelevel of the target mRNA, or inhibition in expression of the targetgene. The pharmaceutical compositions can be used to treat a subject atrisk of developing a disease or disorder that would benefit fromreduction of the level of the target mRNA or an inhibition in expressionthe target gene. In one embodiment, the method includes administering anAAT RNAi agent linked to a targeting ligand as described herein, to asubject to be treated. In some embodiments, one or more pharmaceuticallyacceptable excipients (including vehicles, carriers, diluents, and/ordelivery polymers) are added to the pharmaceutical compositionsincluding an AAT RNAi agent, thereby forming a pharmaceuticalformulation suitable for in vivo delivery to a subject, including ahuman.

The pharmaceutical compositions that include an AAT RNAi agent andmethods disclosed herein decrease the level of the target mRNA in acell, group of cells, group of cells, tissue, or subject, including:administering to the subject a therapeutically effective amount of aherein described AAT RNAi agent, thereby inhibiting the expression ofAAT mRNA in the subject.

In some embodiments, the described pharmaceutical compositions includingan AAT RNAi agent are used for treating or managing clinicalpresentations in a subject with AATD, such as chronic hepatitis,cirrhosis, hepatocellular carcinoma, transaminitis, cholestasis,fibrosis, and even fulminant hepatic failure. In some embodiments, atherapeutically or prophylactically effective amount of one or more ofpharmaceutical compositions is administered to a subject in need of suchtreatment. In some embodiments, administration of any of the disclosedAAT RNAi agents can be used to decrease the number, severity, and/orfrequency of symptoms of a disease in a subject.

The described pharmaceutical compositions including an AAT RNAi agentcan be used to treat at least one symptom in a subject having a diseaseor disorder that would benefit from reduction or inhibition inexpression of AAT mRNA. In some embodiments, the subject is administereda therapeutically effective amount of one or more pharmaceuticalcompositions including an AAT RNAi agent thereby treating the symptom.In other embodiments, the subject is administered a prophylacticallyeffective amount of one or more AAT RNAi agents, thereby preventing theat least one symptom.

The route of administration is the path by which an AAT RNAi agent isbrought into contact with the body. In general, methods of administeringdrugs and nucleic acids for treatment of a mammal are well known in theart and can be applied to administration of the compositions describedherein. The AAT RNAi agents disclosed herein can be administered via anysuitable route in a preparation appropriately tailored to the particularroute. Thus, herein described pharmaceutical compositions can beadministered by injection, for example, intravenously, intramuscularly,intracutaneously, subcutaneously, intraarticularly, orintraperitoneally. In some embodiments, the herein describedpharmaceutical compositions are administered via subcutaneous injection.

The pharmaceutical compositions including an AAT RNAi agent describedherein can be delivered to a cell, group of cells, tissue, or subjectusing oligonucleotide delivery technologies known in the art. Ingeneral, any suitable method recognized in the art for delivering anucleic acid molecule (in vitro or in vivo) can be adapted for use withthe compositions described herein. For example, delivery can be by localadministration, (e.g., direct injection, implantation, or topicaladministering), systemic administration, or subcutaneous, intravenous,intraperitoneal, or parenteral routes, including intracranial (e.g.,intraventricular, intraparenchymal and intrathecal), intramuscular,transdermal, airway (aerosol), nasal, oral, rectal, or topical(including buccal and sublingual) administration. In certainembodiments, the compositions are administered by subcutaneous orintravenous infusion or injection.

Accordingly, in some embodiments, the pharmaceutical compositionsdescribed herein comprise one or more pharmaceutically acceptableexcipients. The pharmaceutical compositions described herein areformulated for administration to a subject.

As used herein, a pharmaceutical composition or medicament includes apharmacologically effective amount of at least one of the described AATRNAi agents and one or more pharmaceutically acceptable excipients.Pharmaceutically acceptable excipients (excipients) are substances otherthan the Active Pharmaceutical Ingredient (API, therapeutic product,e.g., AAT RNAi agent) that are intentionally included in the drugdelivery system. Excipients do not exert or are not intended to exert atherapeutic effect at the intended dosage. Excipients can act to a) aidin processing of the drug delivery system during manufacture, b)protect, support, or enhance stability, bioavailability or patientacceptability of the API, c) assist in product identification, and/or d)enhance any other attribute of the overall safety, effectiveness, ofdelivery of the API during storage or use. A pharmaceutically acceptableexcipient may or may not be an inert substance.

Excipients include, but are not limited to: absorption enhancers,anti-adherents, anti-foaming agents, anti-oxidants, binders, bufferingagents, carriers, coating agents, colors, delivery enhancers, deliverypolymers, dextran, dextrose, diluents, disintegrants, emulsifiers,extenders, fillers, flavors, glidants, humectants, lubricants, oils,polymers, preservatives, saline, salts, solvents, sugars, suspendingagents, sustained release matrices, sweeteners, thickening agents,tonicity agents, vehicles, water-repelling agents, and wetting agents.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, Cremophor®ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Itshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganismssuch as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol,and sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfilter sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Formulations suitable for intra-articular administration can be in theform of a sterile aqueous preparation of the drug that can be inmicrocrystalline form, for example, in the form of an aqueousmicrocrystalline suspension. Liposomal formulations or biodegradablepolymer systems can also be used to present the drug for bothintra-articular and ophthalmic administration.

The active compounds can be prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. Liposomalsuspensions can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

The AAT RNAi agents can be formulated in compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform refers to physically discrete units suited as unitary dosages forthe subject to be treated; each unit containing a predetermined quantityof active compound calculated to produce the desired therapeutic effectin association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of the disclosure are dictatedby and directly dependent on the unique characteristics of the activecompound and the therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

A pharmaceutical composition can contain other additional componentscommonly found in pharmaceutical compositions. Such additionalcomponents include, but are not limited to: anti-pruritics, astringents,local anesthetics, or anti-inflammatory agents (e.g., antihistamine,diphenhydramine, etc.). It is also envisioned that cells, tissues, orisolated organs that express or comprise the herein defined RNAi agentsmay be used as “pharmaceutical compositions.” As used herein,“pharmacologically effective amount,” “therapeutically effectiveamount,” or simply “effective amount” refers to that amount of an RNAiagent to produce a pharmacological, therapeutic or preventive result.

Generally, an effective amount of an active compound will be in therange of from about 0.1 to about 100 mg/kg of body weight/day, e.g.,from about 1.0 to about 50 mg/kg of body weight/day. In someembodiments, an effective amount of an active compound will be in therange of from about 0.25 to about 5 mg/kg of body weight per dose. Insome embodiments, an effective amount of an active ingredient will be inthe range of from about 0.5 to about 4 mg/kg of body weight per dose.The amount administered will also likely depend on such variables as theoverall health status of the patient, the relative biological efficacyof the compound delivered, the formulation of the drug, the presence andtypes of excipients in the formulation, and the route of administration.Also, it is to be understood that the initial dosage administered can,in some instances, be increased beyond the above upper level to rapidlyachieve the desired blood-level or tissue level, or the initial dosagecan, in some instances, be smaller than the optimum.

For treatment of disease or for formation of a medicament or compositionfor treatment of a disease, the pharmaceutical compositions describedherein including an AAT RNAi agent can be combined with an excipient orwith a second therapeutic agent or treatment including, but not limitedto: a second or other RNAi agent, a small molecule drug, an antibody, anantibody fragment, peptide and/or aptamer.

The described AAT RNAi agents, when added to pharmaceutically acceptableexcipients or adjuvants, can be packaged into kits, containers, packs,or dispensers. The pharmaceutical compositions described herein can bepackaged in pre-filled syringes or vials.

Methods of Treatment and Inhibition of Expression

The AAT RNAi agents disclosed herein can be used to treat a subject(e.g., a human or other mammal) having a disease or disorder that wouldbenefit from administration of the compound. In some embodiments, theRNAi agents disclosed herein can be used to treat a subject (e.g., ahuman) having AATD, or symptoms, diseases, or disorders that wouldbenefit from reduction or inhibition in expression of AAT mRNA, such asAATD liver disease. The subject is administered a therapeuticallyeffective amount of any one or more of the AAT RNAi agents describedherein. The subject can be a human, patient, or human patient. Thesubject may be an adult, adolescent, child, or infant. The describedpharmaceutical compositions including an AAT RNAi agent can be used toprovide methods for the therapeutic treatment of diseases, such as AATD.Such methods include administration of a pharmaceutical compositiondescribed herein to a human being or animal.

In some embodiments, the AAT RNAi agents described herein are used totreat a subject with AATD, including symptoms, diseases or disordersrelated to AATD. AATD liver diseases or disorders include, but are notlimited to, chronic hepatitis, cirrhosis, hepatocellular carcinoma,transaminitis, cholestasis, fibrosis, and fulminant hepatic failure.

In some embodiments, the described AAT RNAi agents are used to treat atleast one symptom in a subject having AATD. The subject is administereda therapeutically effective amount of any one or more of the describedRNAi agents.

In certain embodiments, the present invention provides methods fortreatment of AATD in a patient in need thereof, comprising administeringto the patient any of the AAT RNAi agents described herein.

In some embodiments, the AAT RNAi agents are used to treat or manage aclinical presentation of a subject with an AATD liver disease ordisorder. The subject is administered a therapeutically effective amountof one or more of the AAT RNAi agents or AAT RNAi agent-containingcompositions described herein. In some embodiments, the method comprisesadministering a composition comprising an AAT RNAi agent describedherein to a subject to be treated.

In some embodiments, the gene expression level and/or mRNA level of anAAT gene in a subject to whom a described AAT RNAi agent is administeredis reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, orgreater than 99% relative to the subject prior to being administered theAAT RNAi agent or to a subject not receiving the AAT RNAi agent. Thegene expression level and/or mRNA level in the subject is reduced in acell, group of cells, and/or tissue of the subject.

In some embodiments, the protein level of AAT in a subject to whom adescribed AAT RNAi agent has been administered is reduced by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99%relative to the subject prior to being administered the AAT RNAi agentor to a subject not receiving the AAT RNAi agent. The protein level inthe subject is reduced in a cell, group of cells, tissue, blood, and/orother fluid of the subject.

In some embodiments, the Z-AAT polymer protein level in a subject havingAATD to whom a described AAT RNAi agent has been administered is reducedby at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greaterthan 99% relative to the subject prior to being administered the AATRNAi agent or to a subject not receiving the AAT RNAi agent. In someembodiments, the Z-AAT polymer protein level in a subject to whom adescribed AAT RNAi agent has been administered is reduced by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99%relative to the subject prior to being administered the AAT RNAi agentor to a subject not receiving the AAT RNAi agent.

A reduction in AAT gene expression, AAT mRNA, or AAT protein levels canbe assessed and quantified by general methods known in the art. TheExamples disclosed herein forth generally known methods for assessinginhibition of AAT gene expression and reduction in AAT protein levels.The reduction or decrease in AAT mRNA level and/or protein level(including Z-AAT polymer and/or monomer) are collectively referred toherein as a reduction or decrease in AAT or inhibiting or reducing theexpression of AAT.

Cells and Tissues and Non-Human Organisms

Cells, tissues, and non-human organisms that include at least one of theAAT RNAi agents described herein is contemplated. The cell, tissue, ornon-human organism is made by delivering the RNAi agent to the cell,tissue, or non-human organism.

The above provided embodiments and items are now illustrated with thefollowing, non-limiting examples.

EXAMPLES Example 1. Identification of RNAi Agent Sequences and Synthesisof RNAi Agents

A selection process for identifying lead sequences for inhibitingexpression of the AAT gene began with in silico methods to identifyconserved sequences across variants of an AAT gene (SEQ ID NO: 1). TheAAT sequence was initially screened using bioinformatics for19-nucleotide sequences having a complementary sequence in knownvariants of human AAT. Sequences known to have manufacturing challengesand those predicted to have poor RNAi activity based on known parameterswere eliminated. Sequences were then subjected to cross-speciesreactivity analysis to select candidates that would cross-react withcynomolgus monkey AAT. The sequences were also evaluated for specificityto avoid off-target effects against the human and cynomolgus monkeygenomes. One-hundred fifteen (115) sequence families of 19-mers wereselected as candidates.

The duplexes in Table 6 herein were synthesized according to thefollowing procedures:

Synthesis

The sense and antisense strands of the AAT RNAi agents were synthesizedaccording to phosphoramidite technology on solid phase used inoligonucleotide synthesis. Depending on the scale, either a MerMade96E®(Bioautomation) or a MerMade12® (Bioautomation) was used. Syntheses wereperformed on a solid support made of controlled pore glass (CPG, 500 Åor 600 Å, obtained from Prime Synthesis, Aston, Pa., USA). All RNA and2′-modified RNA phosphoramidites were purchased from Thermo FisherScientific (Milwaukee, Wis., USA). Specifically, the following2′-O-methyl phosphoramidites were used:(5′-O-dimethoxytrityl-N6-(benzoyl)-2′-O-methyl-adenosine-3-O-(2-cyanoethyl-N,N-diisopropy-lamino)phosphoramidite,5′-O-dimethoxy-trityl-N4-(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphoramidite,(5′-O-dimethoxytrityl-N2-(isobutyryl)-2′-O-methyl-guanosine-3′-O-(2-cyano-ethyl-N,N-diisopropylamino)phosphoramidite,and5′-O-dimethoxy-trityl-2′-O-methyl-uridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite.The 2′-deoxy-2′-fluoro-phosphoramidites carried the same protectinggroups as the 2′-O-methyl RNA amidites. The following UNAphosphoramidites were used:5′-(4,4′-Dimethoxytrityl)-N-benzoyl-2′,3′-seco-adenosine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphor-amidite,5′-(4,4′-Dimethoxytrityl)-N-acetyl-2′,3′-seco-cytosine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diiso-propyl)]-phosphoramidite,5′-(4,4′-Dimethoxytrityl)-N-isobutyryl-2′,3′-seco-guanosine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and5′-(4,4′-Dimethoxy-trityl)-2′,3′-seco-uridine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diiso-propyl)]-phosphoramidite.

Targeting ligand-containing phosphoramidites were dissolved in anhydrousdichloromethane or anhydrous acetonitrile (50 mM), while all otheramidites were dissolved in anhydrous acetonitrile (50 mM) and molecularsieves (3 Å) were added. 5-Benzylthio-1H-tetrazole (BTT, 250 mM inacetonitrile) or 5-Ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile)was used as activator solution. Coupling times were 10 min (RNA), 15 min(targeting ligand), 90 sec (2′OMe), and 60 sec (2′F). In order tointroduce phosphorothioate linkages, a 100 mM solution of 3-phenyl1,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster,Mass., USA) in anhydrous Acetonitrile was employed.

Cleavage and Deprotection of Support Bound Oligomers.

After finalization of the solid phase synthesis, the dried solid supportwas treated with a 1:1 volume solution of 40 wt. % methylamine in waterand 28% ammonium hydroxide solution (Aldrich) for two hours at 30° C.The solution was evaporated and the solid residue was reconstituted inwater (see below).

Purification

Crude oligomers were purified by anionic exchange HPLC using a TKSgelSuperQ-5PW 13u column and Shimadzu LC-8 system. Buffer A was 20 mM Tris,5 mM EDTA, pH 9.0 and contained 20% Acetonitrile and buffer B was thesame as buffer A with the addition of 1.5 M sodium chloride. UV tracesat 260 nm were recorded. Appropriate fractions were pooled then run onsize exclusion HPLC using a GE Healthcare XK 16/40 column packed withSephadex G-25 medium with a running buffer of 100 mM ammoniumbicarbonate, pH 6.7 and 20% Acetonitrile.

Annealing

Complementary strands were mixed by combining equimolar RNA solutions(sense and antisense) in 0.2×PBS (Phosphate-Buffered Saline, 1×,Corning, Cellgro) to form the RNAi agents. This solution was placed intoa thermomixer at 70° C., heated to 95° C., held at 95° C. for 5 min, andcooled to room temperature slowly. Some RNAi agents were lyophilized andstored at −15 to −25° C. Duplex concentration was determined bymeasuring the solution absorbance on a UV-Vis spectrometer in 0.2×PBS.The solution absorbance at 260 nm was then multiplied by a conversionfactor and the dilution factor to determine the duplex concentration.Unless otherwise stated, all conversion factor was 0.037 mg/(mL·cm). Forsome experiments, a conversion factor was calculated from anexperimentally determined extinction coefficient.

Example 2. In Vitro Testing of AAT RNAi Agents

Candidate sequence duplexes were tested in vitro. The antisense strandsequences and sense strand sequences were annealed to form duplexes of21-mer strands (having 19 base pairs and a di-nucleotide UU overhang oneach 3′ end) for in vitro testing, as shown in the following Table 8:

TABLE 8 Sequences of AAT RNAi Agents in Example 2 SEQ SEQ Duplex IDAntisense Sequence ID Sense Sequence ID NO: (5′ → 3′) NO: (5′ → 3′) No.1035 AGAAGAUAUUGGUGCUGUUUU 1150 AACAGCACCAAUAUCUUCUUU D1 1036AGGAACUUGGUGAUGAUAUUU 1151 AUAUCAUCACCAAGUUCCUUU D2 1037UGUCUUCUGGGCAGCAUCUUU 1152 AGAUGCUGCCCAGAAGACAUU D3 1038UGUUGGACUGGUGUGCCAGUU 1153 CUGGCACACCAGUCCAACAUU D4 1039CUGUUGGACUGGUGUGCCAUU 1154 UGGCACACCAGUCCAACAGUU D5 1040UGCUGUUGGACUGGUGUGCUU 1155 GCACACCAGUCCAACAGCAUU D6 1041UAUUGGUGCUGUUGGACUGUU 1156 CAGUCCAACAGCACCAAUAUU D7 1042AUAUUGGUGCUGUUGGACUUU 1157 AGUCCAACAGCACCAAUAUUU D8 1043GAUAUUGGUGCUGUUGGACUU 1158 GUCCAACAGCACCAAUAUCUU D9 1044AAGAUAUUGGUGCUGUUGGUU 1159 CCAACAGCACCAAUAUCUUUU D10 1045GUAGCGAUGCUCACUGGGGUU 1160 CCCCAGUGAGCAUCGCUACUU D11 1046AAAGGCUGUAGCGAUGCUCUU 1161 GAGCAUCGCUACAGCCUUUUU D12 1047GCAAAGGCUGUAGCGAUGCUU 1162 GCAUCGCUACAGCCUUUGCUU D13 1048UGCAAAGGCUGUAGCGAUGUU 1163 CAUCGCUACAGCCUUUGCAUU D14 1049AUUGCAAAGGCUGUAGCGAUU 1164 UCGCUACAGCCUUUGCAAUUU D15 1050AGCAUUGCAAAGGCUGUAGUU 1165 CUACAGCCUUUGCAAUGCUUU D16 1051AGAGCAUUGCAAAGGCUGUUU 1166 ACAGCCUUUGCAAUGCUCUUU D17 1052GGAGUUCCUGGAAGCCUUCUU 1167 GAAGGCUUCCAGGAACUCCUU D18 1053UCCAAAAACUUAUCCACUAUU 1168 UAGUGGAUAAGUUUUUGGAUU D19 1054AAGGCUUCUGAGUGGUACAUU 1169 UGUACCACUCAGAAGCCUUUU D20 1055GAAGGCUUCUGAGUGGUACUU 1170 GUACCACUCAGAAGCCUUCUU D21 1056UUCUUGGCCUCUUCGGUGUUU 1171 ACACCGAAGAGGCCAAGAAUU D22 1057GUUUCUUGGCCUCUUCGGUUU 1172 ACCGAAGAGGCCAAGAAACUU D23 1058UUGAUCUGUUUCUUGGCCUUU 1173 AGGCCAAGAAACAGAUCAAUU D24 1059GUUGAUCUGUUUCUUGGCCUU 1174 GGCCAAGAAACAGAUCAACUU D25 1060CGUUGAUCUGUUUCUUGGCUU 1175 GCCAAGAAACAGAUCAACGUU D26 1061CACAAUUUUCCCUUGAGUAUU 1176 UACUCAAGGGAAAAUUGUGUU D27 1062UCCACAAUUUUCCCUUGAGUU 1177 CUCAAGGGAAAAUUGUGGAUU D28 1063AUCCACAAUUUUCCCUUGAUU 1178 UCAAGGGAAAAUUGUGGAUUU D29 1064UGUCAAGCUCCUUGACCAAUU 1179 UUGGUCAAGGAGCUUGACAUU D30 1065GUGUCUCUGUCAAGCUCCUUU 1180 AGGAGCUUGACAGAGACACUU D31 1066ACUGUGUCUCUGUCAAGCUUU 1181 AGCUUGACAGAGACACAGUUU D32 1067UGUAAUUCACCAGAGCAAAUU 1182 UUUGCUCUGGUGAAUUACAUU D33 1068UUAAACAUGCCUAAACGCUUU 1183 AGCGUUUAGGCAUGUUUAAUU D34 1069GUUAAACAUGCCUAAACGCUU 1184 GCGUUUAGGCAUGUUUAACUU D35 1070UGUUAAACAUGCCUAAACGUU 1185 CGUUUAGGCAUGUUUAACAUU D36 1071GGAUGUUAAACAUGCCUAAUU 1186 UUAGGCAUGUUUAACAUCCUU D37 1072AUUUCAUCAGCAGCACCCAUU 1187 UGGGUGCUGCUGAUGAAAUUU D38 1073GAAGAAGAUGGCGGUGGCAUU 1188 UGCCACCGCCAUCUUCUUCUU D39 1074GGUGAGUUCAUUUUCCAGGUU 1189 CCUGGAAAAUGAACUCACCUU D40 1075GAACUUGGUGAUGAUAUCGUU 1190 CGAUAUCAUCACCAAGUUCUU D41 1076CAUUUUCCAGGAACUUGGUUU 1191 ACCAAGUUCCUGGAAAAUGUU D42 1077CAUAGGUUCCAGUAAUGGAUU 1192 UCCAUUACUGGAACCUAUGUU D43 1078UCAUAGGUUCCAGUAAUGGUU 1193 CCAUUACUGGAACCUAUGAUU D44 1079UCAGAUCAUAGGUUCCAGUUU 1194 ACUGGAACCUAUGAUCUGAUU D45 1080UCUUCAGAUCAUAGGUUCCUU 1195 GGAACCUAUGAUCUGAAGAUU D46 1081CUCUUCAGAUCAUAGGUUCUU 1196 GAACCUAUGAUCUGAAGAGUU D47 1082GAGGUCAGCCCCAUUGCUGUU 1197 CAGCAAUGGGGCUGACCUCUU D48 1083GAGAGGUCAGCCCCAUUGCUU 1198 GCAAUGGGGCUGACCUCUCUU D49 1084CUUCAGGGGUGCCUCCUCUUU 1199 AGAGGAGGCACCCCUGAAGUU D50 1085GAGAGCUUCAGGGGUGCCUUU 1200 AGGCACCCCUGAAGCUCUCUU D51 1086UUAUGCACGGCCUUGGAGAUU 1201 UCUCCAAGGCCGUGCAUAAUU D52 1087CCUUAUGCACGGCCUUGGAUU 1202 UCCAAGGCCGUGCAUAAGGUU D53 1088GCCUUAUGCACGGCCUUGGUU 1203 CCAAGGCCGUGCAUAAGGCUU D54 1089AGCCUUAUGCACGGCCUUGUU 1204 CAAGGCCGUGCAUAAGGCUUU D55 1090CGAUGGUCAGCACAGCCUUUU 1205 AAGGCUGUGCUGACCAUCGUU D56 1091GUCGAUGGUCAGCACAGCCUU 1206 GGCUGUGCUGACCAUCGACUU D57 1092AAAAACAUGGCCCCAGCAGUU 1207 CUGCUGGGGCCAUGUUUUUUU D58 1093CUAAAAACAUGGCCCCAGCUU 1208 GCUGGGGCCAUGUUUUUAGUU D59 1094UCUAAAAACAUGGCCCCAGUU 1209 CUGGGGCCAUGUUUUUAGAUU D60 1095CCUCUAAAAACAUGGCCCCUU 1210 GGGGCCAUGUUUUUAGAGGUU D61 1096GCCUCUAAAAACAUGGCCCUU 1211 GGGCCAUGUUUUUAGAGGCUU D62 1097UAGACAUGGGUAUGGCCUCUU 1212 GAGGCCAUACCCAUGUCUAUU D63 1098GAUAGACAUGGGUAUGGCCUU 1213 GGCCAUACCCAUGUCUAUCUU D64 1099UGUUGAACUUGACCUCGGGUU 1214 CCCGAGGUCAAGUUCAACAUU D65 1100GGUUUGUUGAACUUGACCUUU 1215 AGGUCAAGUUCAACAAACCUU D66 1101AAAGGGUUUGUUGAACUUGUU 1216 CAAGUUCAACAAACCCUUUUU D67 1102ACAAAGGGUUUGUUGAACUUU 1217 AGUUCAACAAACCCUUUGUUU D68 1103GACAAAGGGUUUGUUGAACUU 1218 GUUCAACAAACCCUUUGUCUU D69 1104AAGACAAAGGGUUUGUUGAUU 1219 UCAACAAACCCUUUGUCUUUU D70 1105CAUUAAGAAGACAAAGGGUUU 1220 ACCCUUUGUCUUCUUAAUGUU D71 1106AUCAUUAAGAAGACAAAGGUU 1221 CCUUUGUCUUCUUAAUGAUUU D72 1107GAAGAGGGGAGACUUGGUAUU 1222 UACCAAGUCUCCCCUCUUCUU D73 1108CCAUGAAGAGGGGAGACUUUU 1223 AAGUCUCCCCUCUUCAUGGUU D74 1109CCCAUGAAGAGGGGAGACUUU 1224 AGUCUCCCCUCUUCAUGGGUU D75 1110UUCCCAUGAAGAGGGGAGAUU 1225 UCUCCCCUCUUCAUGGGAAUU D76 1111UUUCCCAUGAAGAGGGGAGUU 1226 CUCCCCUCUUCAUGGGAAAUU D77 1112AACCCUUCUUUAAUGUCAUUU 1227 AUGACAUUAAAGAAGGGUUUU D78 1113UUGUUGGACUGGUGUGCCAUU 1228 UGGCACACCAGUCCAACAAUU D79 1114UAUAUUGGUGCUGUUGGACUU 1229 GUCCAACAGCACCAAUAUAUU D80 1115UUAGCGAUGCUCACUGGGGUU 1230 CCCCAGUGAGCAUCGCUAAUU D81 1116UCAAAGGCUGUAGCGAUGCUU 1231 GCAUCGCUACAGCCUUUGAUU D82 1117UGAGUUCCUGGAAGCCUUCUU 1232 GAAGGCUUCCAGGAACUCAUU D83 1118UAAGGCUUCUGAGUGGUACUU 1233 GUACCACUCAGAAGCCUUAUU D84 1119UUUUCUUGGCCUCUUCGGUUU 1234 ACCGAAGAGGCCAAGAAAAUU D85 1120UUUGAUCUGUUUCUUGGCCUU 1235 GGCCAAGAAACAGAUCAAAUU D86 1121UGUUGAUCUGUUUCUUGGCUU 1236 GCCAAGAAACAGAUCAACAUU D87 1122UACAAUUUUCCCUUGAGUAUU 1237 UACUCAAGGGAAAAUUGUAUU D88 1123UUGUCUCUGUCAAGCUCCUUU 1238 AGGAGCUUGACAGAGACAAUU D89 1124UUUAAACAUGCCUAAACGCUU 1239 GCGUUUAGGCAUGUUUAAAUU D90 1125UGAUGUUAAACAUGCCUAAUU 1240 UUAGGCAUGUUUAACAUCAUU D91 1126UAAGAAGAUGGCGGUGGCAUU 1241 UGCCACCGCCAUCUUCUUAUU D92 1127UGUGAGUUCAUUUUCCAGGUU 1242 CCUGGAAAAUGAACUCACAUU D93 1128UAACUUGGUGAUGAUAUCGUU 1243 CGAUAUCAUCACCAAGUUAUU D94 1129UAUUUUCCAGGAACUUGGUUU 1244 ACCAAGUUCCUGGAAAAUAUU D95 1130UAUAGGUUCCAGUAAUGGAUU 1245 UCCAUUACUGGAACCUAUAUU D96 1131UUCUUCAGAUCAUAGGUUCUU 1246 GAACCUAUGAUCUGAAGAAUU D97 1132UAGGUCAGCCCCAUUGCUGUU 1247 CAGCAAUGGGGCUGACCUAUU D98 1133UAGAGGUCAGCCCCAUUGCUU 1248 GCAAUGGGGCUGACCUCUAUU D99 1134UUUCAGGGGUGCCUCCUCUUU 1249 AGAGGAGGCACCCCUGAAAUU D100 1135UAGAGCUUCAGGGGUGCCUUU 1250 AGGCACCCCUGAAGCUCUAUU D101 1136UCUUAUGCACGGCCUUGGAUU 1251 UCCAAGGCCGUGCAUAAGAUU D102 1137UCCUUAUGCACGGCCUUGGUU 1252 CCAAGGCCGUGCAUAAGGAUU D103 1138UGAUGGUCAGCACAGCCUUUU 1253 AAGGCUGUGCUGACCAUCAUU D104 1139UUCGAUGGUCAGCACAGCCUU 1254 GGCUGUGCUGACCAUCGAAUU D105 1140UUAAAAACAUGGCCCCAGCUU 1255 GCUGGGGCCAUGUUUUUAAUU D106 1141UCUCUAAAAACAUGGCCCCUU 1256 GGGGCCAUGUUUUUAGAGAUU D107 1142UCCUCUAAAAACAUGGCCCUU 1257 GGGCCAUGUUUUUAGAGGAUU D108 1143UAUAGACAUGGGUAUGGCCUU 1258 GGCCAUACCCAUGUCUAUAUU D109 1144UGUUUGUUGAACUUGACCUUU 1259 AGGUCAAGUUCAACAAACAUU D110 1145UACAAAGGGUUUGUUGAACUU 1260 GUUCAACAAACCCUUUGUAUU D111 1146UAUUAAGAAGACAAAGGGUUU 1261 ACCCUUUGUCUUCUUAAUAUU D112 1147UAAGAGGGGAGACUUGGUAUU 1262 UACCAAGUCUCCCCUCUUAUU D113 1148UCAUGAAGAGGGGAGACUUUU 1263 AAGUCUCCCCUCUUCAUGAUU D114 1149UCCAUGAAGAGGGGAGACUUU 1264 AGUCUCCCCUCUUCAUGGAUU D115

AAT RNAi agents were evaluated by transfection of Hep3B cells, a humanhepatocellular carcinoma line. Cells were plated at −10,000 cells perwell in 96-well format, and each of the 115 AAT RNAi agent duplexes wastransfected at three concentrations (10 nM, 1 nM, and 0.1 nM), usingLipoFectamine RNAiMax (Thermo Fisher) transfection reagent. Relativeexpression of each of the 115 AAT RNAi agents was determined by qRT-PCRby comparing the expression levels of AAT mRNA to an endogenous control,and normalized to untreated Hep3B cells (ΔΔC_(T) analysis), as shown inTable 9.

TABLE 9 In Vitro Data from Duplexes of Example 2 Duplex ID No. Avg. Rel.Avg. Rel. Avg. Rel. (From Table 8) Exp. 10 nM Exp. 1 nM Exp. 0.1 nM D11.037 0.896 0.709 D2 0.068 0.089 0.381 D3 0.046 0.064 0.403 D4 0.0750.090 0.391 D5 0.408 0.424 0.743 D6 0.018 0.032 0.347 D7 0.069 0.1250.666 D8 0.092 0.193 0.794 D9 0.206 0.228 0.839 D10 0.023 0.032 0.235D11 0.309 0.522 0.894 D12 0.049 0.092 0.732 D13 0.549 0.665 0.955 D140.531 0.654 0.934 D15 0.108 0.197 0.820 D16 0.558 0.516 0.834 D17 0.6260.606 0.841 D18 0.668 0.703 0.778 D19 0.624 0.803 0.785 D20 0.071 0.0800.506 D21 0.022 0.037 0.345 D22 0.086 0.127 0.588 D23 0.175 0.238 0.893D24 0.134 0.078 0.368 D25 0.056 0.075 0.687 D26 0.122 0.196 0.756 D270.517 0.560 0.846 D28 0.801 0.838 0.884 D29 0.820 0.870 0.903 D30 0.5580.632 0.879 D31 1.112 1.110 0.922 D32 0.246 0.359 1.041 D33 0.107 0.3550.967 D34 0.096 0.170 0.962 D35 0.317 0.552 0.949 D36 0.064 0.134 0.873D37 0.463 1.005 1.006 D38 0.428 0.688 0.486 D39 0.730 0.918 1.258 D400.059 0.067 0.912 D41 0.093 0.095 0.952 D42 0.582 0.665 0.944 D43 0.1960.283 1.004 D44 0.195 0.278 0.860 D45 0.053 0.103 0.817 D46 0.082 0.1271.034 D47 0.089 0.156 0.821 D48 0.735 0.695 0.838 D49 0.604 0.610 0.838D50 0.543 0.633 0.806 D51 0.114 0.144 0.775 D52 0.108 0.203 0.836 D531.062 0.836 0.931 D54 0.091 0.274 1.081 D55 0.526 0.623 0.914 D56 0.5000.588 0.884 D57 0.049 0.126 0.797 D58 0.198 0.302 0.917 D59 0.732 0.7450.953 D60 0.389 0.580 0.897 D61 0.585 0.624 1.802 D62 0.174 0.215 1.115D63 0.093 0.074 0.917 D64 0.133 0.133 1.055 D65 0.395 0.362 0.986 D660.054 0.055 1.083 D67 0.105 0.118 1.018 D68 0.106 0.122 1.290 D69 0.2010.194 1.062 D70 0.050 0.048 0.709 D71 0.231 0.216 0.767 D72 0.046 0.0300.737 D73 0.521 0.423 0.782 D74 0.479 0.467 0.694 D75 0.531 0.583 0.794D76 0.210 0.285 0.924 D77 0.152 0.181 0.803 D78 0.425 0.485 0.703 D790.120 0.127 0.711 D80 0.203 0.167 0.672 D81 0.477 0.402 0.611 D82 0.5400.489 0.661 D83 0.315 0.316 0.838 D84 0.135 0.118 0.375 D85 0.209 0.2701.050 D86 0.120 0.136 0.928 D87 0.172 0.207 1.056 D88 0.218 0.308 1.006D89 0.605 0.643 0.925 D90 0.205 0.259 0.927 D91 0.594 1.097 1.052 D920.337 0.887 1.015 D93 0.068 0.503 0.864 D94 0.067 0.475 0.811 D95 0.1860.770 0.931 D96 0.062 0.389 0.550 D97 0.066 0.470 0.896 D98 0.567 0.9981.044 D99 0.451 1.092 1.359 D100 0.292 0.745 0.875 D101 0.049 0.3200.659 D102 0.313 0.799 0.732 D103 0.068 0.541 0.630 D104 0.077 0.5520.682 D105 0.071 0.355 0.459 D106 1.179 1.117 1.076 D107 0.328 0.5970.876 D108 0.125 0.467 0.573 D109 0.141 0.545 0.753 D110 0.076 0.4970.778 D111 0.132 0.511 0.634 D112 0.216 0.586 0.784 D113 0.462 0.6871.021 D114 0.507 0.792 1.170 D115 0.259 0.797 1.027

Example 3. In Vivo Testing of NAG-Conjugated AAT RNAi Agents in PiZ Mice

A transgenic PiZ mouse model (PiZ mice) was used to evaluate AAT RNAiagents in vivo. PiZ mice harbor the human PiZ AAT mutant allele andmodel human AATD (Carlson et al., Journal of Clinical Investigation1989).

NAG-conjugated AAT RNAi agents were prepared in a pharmaceuticallyacceptable saline buffer and administered to PiZ mice to evaluateknockdown of AAT gene expression. On day 1, each mouse received a singlesubcutaneous (SQ) dose into the loose skin on the back between theshoulders of 5.0 mg/kg (mpk) of either AD04446, AD04447, AD04448,AD04449, AD04450, AD04451, AD04454, AD04455, AD04456, AD04457, AD04458,or AD04459. (See Tables 4-7 for the modified AAT RNAi agents and NAGligand structures). AAT RNAi agents AD04451 and AD04459 included amodified nucleotide antisense strand sequence designed to target an AATgene (SEQ ID NO: 1) at position 1000; AAT RNAi agents AD04446 andAD04454 included a modified nucleotide antisense strand sequencedesigned to target an AAT gene (SEQ ID NO: 1) at position 1142; AAT RNAiagents AD04447 and AD04455 included a modified nucleotide antisensestrand sequence designed to target an AAT gene (SEQ ID NO: 1) atposition 1211; AAT RNAi agents AD04448 and AD04456 included a modifiednucleotide antisense strand sequence designed to target an AAT gene (SEQID NO: 1) at position 1326; AAT RNAi agents AD04449 and AD04457 includeda modified nucleotide antisense strand sequence designed to target anAAT gene (SEQ ID NO: 1) at position 1338; and AAT RNAi agents AD04450and AD04458 included a modified nucleotide antisense strand sequencedesigned to target an AAT gene (SEQ ID NO: 1) at position 1427. (Seealso Tables 1 and 2). Three mice were dosed with each AAT RNAi agent(n=3).

Plasma samples were drawn and analyzed for AAT (Z-AAT) protein levels onday 1 (pre-dose), day 8, day 15, day 22, day 29, and day 36. AAT levelswere normalized to day 1 (pre-dose) AAT plasma levels. Protein levelswere measured by quantifying circulating human Z-AAT levels in plasma bya commercially available ELISA kit according to the manufacturer'srecommendations. The average normalized AAT (Z-AAT) levels for each RNAiagent are reported in the following Table 10:

TABLE 10 Average Normalized AAT Protein (Normalized to Pre-Treatment)from Example 3 Day 8 Day 15 Day 22 Day 29 Day 36 Avg Std Dev Avg Std DevAvg Std Dev Avg Std Dev Avg Std Dev Group ID AAT (+/−) AAT (+/−) AAT(+/−) AAT (+/−) AAT (+/−) Group 1 (5.0 1.010 0.256 1.050 0.108 1.4510.137 1.145 0.154 1.117 0.080 mg/kg AD04447) Group 2 (5.0 0.884 0.2620.866 0.276 1.306 0.112 1.147 0.119 1.076 0.172 mg/kg AD04448) Group 3(5.0 0.909 0.060 0.969 0.152 1.290 0.185 1.290 0.201 1.245 0.106 mg/kgAD04449) Group 4 (5.0 0.595 0.083 0.799 0.131 1.099 0.256 1.090 0.3461.229 0.444 mg/kg AD04450) Group 5 (5.0 0.282 0.006 0.525 0.020 1.3580.188 1.767 0.325 1.586 0.297 mg/kg AD04451) Group 6 (5.0 0.656 0.1260.639 0.039 0.741 0.089 0.738 0.235 0.819 0.156 mg/kg AD04455) Group 7(5.0 0.605 0.129 0.469 0.036 0.717 0.105 0.662 0.097 0.875 0.195 mg/kgAD04456) Group 8 (5.0 0.501 0.108 0.663 0.091 1.031 0.324 1.176 0.3681.603 0.597 mg/kg AD04457) Group 9 (5.0 0.308 0.081 0.174 0.031 0.1770.010 0.211 0.010 0.345 0.041 mg/kg AD04458) Group 10 (5.0 0.256 0.0210.134 0.045 0.174 0.084 0.234 0.174 0.315 0.336 mg/kg AD04459) Group 11(5.0 0.686 0.178 0.739 0.130 0.973 0.263 0.955 0.107 0.885 0.119 mg/kgAD04446) Group 12 (5.0 0.338 0.014 0.361 0.105 0.602 0.252 0.729 0.2660.970 0.245 mg/kg AD04454)

As shown from the data in Table 10, above, while AAT RNAi agent AD04447showed essentially no reduction in AAT protein, AAT RNAi agents AD04458(which included a modified nucleotide sequence designed to target an AATgene (SEQ ID NO: 1) at position 1427) and AD04459 (which included amodified nucleotide sequence designed to target an AAT gene (SEQ IDNO: 1) at position 1000) showed a substantial reduction in AAT proteinacross all timepoints. For example, AD04458 showed knockdown ofapproximately 69% at day 8 (0.308); approximately 83% at day 15 (0.174),and approximately 82% at day 22 (0.177). Additionally, for example,AD04459 showed a knockdown of approximately 74% at day 8 (0.256),approximately 87% at day 15 (0.134), and approximately 83% at day 22(0.174).

Example 4. In Vivo Testing of NAG-Conjugated AAT RNAi Agents inCynomolgus Monkeys

NAG-conjugated AAT RNAi agents were made and combined in apharmaceutically acceptable saline buffer as known in the art forsubcutaneous (SC) injection. On day 1, cynomolgus macaque (Macacafascicularis) primates (referred to herein as “cynos” or “monkeys”) wereinjected subcutaneously with 3 mg/kg of either AD04824, AD04825,AD04826, or AD04827 (see Tables 4-7 for the modified AAT RNAi agents andNAG ligand structures). Each of these AAT RNAi agents included amodified nucleotide sequence designed to target an AAT gene (SEQ IDNO: 1) at position 1000, and was cross-reactive with cynos. Threemonkeys in each group were tested (n=3).

Serum samples from treated cynomolgus monkeys were taken on day −7 andday 1 (pre-dose), and on days 8, 15, 22, and 29 to monitor knockdown.Day 36 was also measured for cynos that were injected with AD04825 andAD04826. At the indicated time points, blood samples were drawn andanalyzed for cynomolgus monkey AAT (cAAT). Blood was collected from thefemoral vein. cAAT levels were determined on a Cobas Integra 400 Plus(Roche Diagnostics) according to the manufacturer's recommendations. AATlevels for each animal at a respective time point was divided by thepre-treatment level (average of day −7 and day 1 (pre-dose)) ofexpression in that animal to determine the ratio of expression“normalized to pre-dose.”

Normalized cynomolgus monkey AAT (cAAT) protein levels after treatmentwith each respective AAT RNAi agent are reported in the following Table11:

TABLE 11 Normalized cAAT Protein (Normalized to Pre-Treatment) fromExample 4 in Cynomolgus Monkeys. Day 8 Day 15 Day 22 Day 29 Day 36 GroupID cAAT cAAT cAAT cAAT cAAT Group 1, Cyno A 0.62 0.52 0.45 0.52 (3.0mg/kg AD04824) Group 1, Cyno B 0.60 0.36 0.32 0.32 (3.0 mg/kg AD04824)Group 1, Cyno C 0.62 0.44 0.41 0.41 (3.0 mg/kg AD04824) Group 2, Cyno A0.58 0.33 0.24 0.24 0.22 (3.0 mg/kg AD04825) Group 2, Cyno B 0.58 0.380.27 0.25 0.27 (3.0 mg/kg AD04825) Group 2, Cyno C 0.79 0.58 0.43 0.430.44 (3.0 mg/kg AD04825) Group 3, Cyno A 0.75 0.59 0.44 0.42 0.38 (3.0mg/kg AD04826) Group 3, Cyno B 0.66 0.43 0.30 0.26 0.24 (3.0 mg/kgAD04826) Group 3, Cyno C 0.62 0.36 0.27 0.25 0.25 (3.0 mg/kg AD04826)Group 4, Cyno A 0.57 0.38 0.26 0.26 (3.0 mg/kg AD04827) Group 4, Cyno B0.61 0.37 0.34 0.34 (3.0 mg/kg AD04827) Group 4, Cyno C 0.66 0.43 0.410.39 (3.0 mg/kg AD04827)

Average normalized cAAT levels for each of the respective treatmentgroups is shown in the bar graph of FIG. 9. As illustrated in Table 11,above, and in FIG. 9, each of the AAT RNAi agents tested showedsubstantial knockdown of cAAT in cynomolgus monkeys across all timepoints measured.

Example 5. In Vivo Testing of NAG-Conjugated AAT RNAi Agents inCynomolgus Monkeys

NAG-conjugated AAT RNAi agents were made and combined in apharmaceutically acceptable saline buffer as known in the art forsubcutaneous (SC) injection. On day 1, cynomolgus macaque (Macacafascicularis) primates were injected subcutaneously with 3 mg/kg ofeither AD04828, AD04831, AD04836, or AD04837 (see Tables 4-7 for themodified AAT RNAi agents and NAG ligand structures). Each of these AATRNAi agents included a modified nucleotide sequence designed to targetan AAT gene (SEQ ID NO: 1) at position 1000, and was cross-reactive withcynos. Three monkeys in each group were tested (n=3) for AD04828 andAD04831, and two monkeys in each group were tested (n=2) for AD04836 andAD04837.

Serum samples from treated cynos were taken on day −35 and day 1(pre-dose), and on days 8, 15, 21, and 29 to monitor knockdown. At theindicated time points, blood samples were drawn and analyzed for cAAT.Blood was collected from the femoral vein. cAAT levels were determinedon a Cobas Integra 400 Plus (Roche Diagnostics) according to themanufacturer's recommendations. cAAT levels for each animal at arespective time point was divided by the pre-treatment level (average ofday −35 and day 1 (pre-dose)) of expression in that animal to determinethe ratio of expression “normalized to pre-treatment”.

Normalized cynomolgus monkey AAT (cAAT) protein levels after treatmentwith each respective AAT RNAi agent are reported in the following Table12:

TABLE 12 Normalized AAT Protein (Normalized to Pre-Treatment) fromExample 5 in Cynomolgus Monkeys Day 8 Day 15 Day 22 Day 29 Group ID cAATcAAT cAAT cAAT Group 1, Cyno A (3.0 mg/kg AD04828) 0.60 0.32 0.25 0.23Group 1, Cyno B (3.0 mg/kg AD04828) 0.67 0.59 0.61 0.76 Group 1, Cyno C(3.0 mg/kg AD04828) 0.51 0.35 0.29 0.29 Group 2, Cyno A (3.0 mg/kgAD04831) 0.68 0.43 0.32 0.28 Group 2, Cyno B (3.0 mg/kg AD04831) 0.710.49 0.47 0.44 Group 2, Cyno C (3.0 mg/kg AD04831) 0.61 0.43 0.34 0.30Group 3, Cyno A (3.0 mg/kg AD04836) 0.61 0.37 0.27 0.23 Group 3, Cyno B(3.0 mg/kg AD04836) 0.67 0.43 0.32 0.27 Group 4, Cyno A (3.0 mg/kgAD04837) 0.65 0.40 0.28 0.24 Group 4, Cyno B (3.0 mg/kg AD04837) 0.550.29 0.20 0.17

Average normalized cAAT levels for each of the respective treatmentgroups is shown in the bar graph of FIG. 10. As shown above in Table 12,as well as in the bar graph of FIG. 10, each of the AAT RNAi agentstested showed substantial knockdown of cAAT in cynomolgus monkeys acrossall time points measured.

Example 6. In Vivo Testing of NAG-Conjugated AAT RNAi Agents in PiZ Mice

The transgenic PiZ mouse model (PiZ mice) as set forth in Example 3 wasused to evaluate AAT RNAi agents in vivo. NAG-conjugated AAT RNAi agentswere prepared in a pharmaceutically acceptable saline buffer andadministered to PiZ mice to evaluate knockdown of AAT gene expression.On day 1, each mouse received a single subcutaneous (SQ) dose into theloose skin on the back between the shoulders of 2.0 mg/kg (mpk) ofeither AD04824, AD04828, AD04829, AD04830, AD04831, AD04832, AD04833,AD04834, AD04836, AD04837, AD04838, AD04839, or AD04857. (See Tables 4-7for the modified AAT RNAi agents and NAG ligand structures). Each of theAAT RNAi agents in this study included a modified nucleotide antisensestrand sequence designed to target an AAT gene (SEQ ID NO: 1) atposition 1000. (See also Tables 1 and 2). Three mice were dosed witheach AAT RNAi agent (n=3).

Plasma samples were drawn and analyzed for AAT (Z-AAT) protein levels ondays −2, day 1 (pre-dose), day 8, day 15, day 22, day 29, and day 36.AAT levels were normalized to day 1 (pre-dose) AAT plasma levels.Protein levels were measured by quantifying circulating human Z-AATlevels in plasma by a commercially available ELISA kit according to themanufacturer's recommendations. The average normalized AAT (Z-AAT)levels for each RNAi agent are reported in the following Table 13:

TABLE 13 Average Normalized AAT Protein (Normalized to Pre-Treatment)from Example 6 Day 8 Day 15 Day 22 Day 29 Day 36 Avg Std Dev Avg Std DevAvg Std Dev Avg Std Dev Avg Std Dev Group ID AAT (+/−) AAT (+/−) AAT(+/−) AAT (+/−) AAT (+/−) Group 1 (2.0 0.105 0.036 0.140 0.067 0.2040.108 0.313 0.104 0.437 0.229 mg/kg AD04824) Group 2 (2.0 0.141 0.0550.236 0.111 0.304 0.138 0.624 0.289 0.814 0.139 mg/kg AD04828) Group 3(2.0 0.109 0.072 0.119 0.102 0.140 0.119 0.141 0.116 0.179 0.145 mg/kgAD04829) Group 4 (2.0 0.147 0.095 0.190 0.148 0.307 0.192 0.521 0.4240.547 0.202 mg/kg AD04830) Group 5 (2.0 0.154 0.104 0.215 0.171 0.4490.375 0.701 0.519 0.584 0.418 mg/kg AD04831) Group 6 (2.0 0.088 0.0320.089 0.048 0.090 0.046 0.117 0.071 0.193 0.131 mg/kg AD04832) Group 7(2.0 0.168 0.029 0.282 0.047 0.448 0.048 0.748 0.223 1.361 0.346 mg/kgAD04833) Group 8 (2.0 0.159 0.037 0.255 0.159 0.470 0.315 0.662 0.3460.728 0.141 mg/kg AD04834) Group 9 (2.0 0.108 0.035 0.070 0.024 0.0830.032 0.090 0.035 0.168 0.078 mg/kg AD04836) Group 10 (2.0 0.157 0.0710.209 0.104 0.242 0.097 0.417 0.198 0.550 0.193 mg/kg AD04837) Group 11(2.0 0.106 0.017 0.099 0.022 0.108 0.039 0.158 0.072 0.188 0.050 mg/kgAD04838) Group 12 (2.0 0.096 0.026 0.069 0.019 0.089 0.036 0.120 0.0380.186 0.083 mg/kg AD04839) Group 12 (2.0 0.272 0.130 0.302 0.145 0.4780.187 0.815 0.436 1.772 1.412 mg/kg AD04857)

As shown from the data in Table 13, above, each of the AAT RNAi agentshowed a substantial reduction in AAT protein through at least day 29.For example, at day 15, each of the AAT RNAi agents tested achieved atleast approximately 70% knockdown of protein compared to pre-treatmentlevels, with multiple groups achieving 90% or better knockdown.

Example 7. In Vivo Testing of NAG-Conjugated AAT RNAi Agents in PiZ Mice

The transgenic PiZ mouse model described in Example 3 was used. Eachmouse was 5 weeks old at the beginning of the study. NAG-conjugated AATRNAi agents were prepared in a pharmaceutically acceptable saline bufferand administered to PiZ mice to evaluate knockdown of AAT geneexpression. Starting on day 1, each mouse received a subcutaneous (SQ)dose q2w (i.e., one injection every two weeks, for a total of 4injections) into the loose skin on the back between the shoulders of 4.0mg/kg (mpk) of either: (1) saline vehicle; (2) AD04837 (see Tables 4-7for the modified AAT RNAi agent and NAG ligand structures), which asnoted previously included a modified nucleotide antisense strandsequence designed to target an AAT gene (SEQ ID NO: 1) at position 1000;or (3) a NAG-conjugated RNAi agent that included a nucleotide sequencetargeting the HBV gene, to be used as a negative control. Singlesubcutaneous injections for the saline vehicle group, AAT RNAi agentgroup, and HBV RNAi agent group were performed on days 1, 15, 29, and43. Seven (7) mice were dosed q2w with the saline vehicle (Group 1);nine (9) mice were dosed q2w with the AAT RNAi agent (Group 2); and six(6) mice were dosed with the HBV RNAi agent (Group 3). The mice in thethree treatment groups were sacrificed on day 57 (13 weeks old). Inaddition to the treatment groups, seven (7) mice were sacrificed at week1 of the study (i.e., 5-week old mice) to serve as a baseline control.

Plasma samples were drawn and analyzed for AAT (Z-AAT) protein levels onday 1 (pre-dose), day 8, day 15, day 22, day 29, and day 36 for allgroups. Additional samples for the AAT RNAi agent group and the salinevehicle group were drawn on day 43, day 50, and day 57. AAT levels werenormalized to day 1 (pre-dose) AAT plasma levels. Protein levels weremeasured by quantifying circulating human Z-AAT levels in plasma by acommercially available ELISA kit according to the manufacturer'srecommendations. The average normalized AAT (Z-AAT) levels for thesaline vehicle and each RNAi agent are reported in the following Table14:

TABLE 14 Average Normalized AAT Protein (Normalized to Pre-Treatment)from Example 7 Day 8 Day 15 Day 22 Day 29 Avg Std Dev Avg Std Dev AvgStd Dev Avg Std Dev Group ID AAT (+/−) AAT (+/−) AAT (+/−) AAT (+/−)Group 1 (saline vehicle) (n = 7) 0.876 0.172 1.264 0.386 1.234 0.4571.319 0.453 Group 2 (AD04837) (n = 9) 0.139 0.050 0.146 0.064 0.0670.029 0.072 0.038 Group 3 (HBV RNAi agent − negative 1.212 0.360 1.0190.201 1.540 0.155 1.585 0.640 control) (n = 6) Day 36 Day 43 Day 50 Day57 Avg Std Dev Avg Std Dev Avg Std Dev Avg Std Dev Group ID AAT (+/−)AAT (+/−) AAT (+/−) AAT (+/−) Group 1 (saline vehicle) (n = 7) 1.2670.491 1.441 0.416 1.172 0.340 1.058 0.299 Group 2 (AD04837) (n = 9)0.040 0.011 0.051 0.020 0.034 0.007 0.038 0.009 Group 3 (HBV RNAi agent− negative 1.665 0.476 1.943 0.221 1.580 0.491 2.001 0.770 control) (n =6)

As shown from the data in Table 14, above, the HBV RNAi agent performedsuccessfully as a negative control showing essentially no AATinhibition. Further, the NAG-conjugated AAT RNAi agent (AD04837)achieved significant knockdown of expression compared to saline and theHBV RNAi agent negative control across all timepoints. In dosing q2w,the AAT RNAi agent in Example 7 showed a knockdown of approximately 96%of AAT protein at day 36 (0.040) and maintained a similar level ofknockdown through day 57.

In addition to monitoring serum AAT levels, homogenized liver tissuefrom PiZ mice treated with NAG-conjugated AAT RNAi agent (AD04837) wasfurther analyzed to determine if both soluble Z-AAT (which is expectedto be predominantly monomeric protein), and insoluble polymers of Z-AAT(which is expected to be polymeric protein) were effectively reduced. Amodified western blot protocol was used to separate the soluble andinsoluble Z-AAT fractions under non-denaturing conditions as previouslydescribed and known in the art (see, e.g., Mueller et al., MolecularTherapy, March 2012, 20(3): 590-600).

A western blot was prepared to examine certain livers of the sacrificedmice. Specifically, livers were examined of (i) 6 baseline mice; (ii) 5AAT RNAi agent mice; and (iii) 4 saline mice. (The gels used for thewestern blot analysis included 15 wells). The samples for the animalsused for this western blot were randomly selected from the variousgroups. FIGS. 11 and 12 show bar graphs reflecting the Z-AAT polymer andZ-AAT monomer levels quantified from the western blot analysis.

As seen from the bar graph in FIG. 11, which reports the monomericprotein levels, when compared to baseline each of the mice dosed withAAT RNAi agent shown a significant reduction in AAT monomeric proteinacross all time points, indicating significant inhibition of the gene.Further, as shown in FIG. 12, which reports the polymeric proteinlevels, the animals treated with the saline vehicle continued to haveincreased polymeric AAT burden after 8 weeks. Conversely, the animalstreated with AAT RNAi agent showed a reduction in polymeric burden ofapproximately 50% over the course of 8 weeks as compared to the baseline(5-week-old) mice, indicating that the administration of NAG-conjugatedAAT RNAi agent (AD04837) is capable of preventing and potentiallyreversing the production of polymeric AAT protein.

Example 8. In Vivo Testing of NAG-Conjugated AAT RNAi Agents in PiZ Mice

The transgenic PiZ mouse model described in Example 3 was used toevaluate RNAi agents in vivo. Each mouse received a single subcutaneous(SQ) dose on day 1 into the loose skin on the back between the shouldersof either: (1) saline; (2) 1.0 mg/kg of the NAG-conjugated AAT RNAiagent of AD04837 (which includes a modified nucleotide antisense strandsequence designed to target an AAT gene (SEQ ID NO: 1) at position1000); (3) 2.0 mg/kg of AD04837; (4) 4.0 mg/kg of AD04837; or (5) 8.0mg/kg of AD04837. Four animals were dosed in group 1 (saline), and allfour were sacrificed on day 43. Fifteen (15) animals were dosed in eachof groups 2, 3, 4, and 5, and 3 animals from each group were sacrificedon day 8, day 15, day 22, day 29, and day 43, respectively.

Plasma samples were drawn and analyzed for AAT (Z-AAT) protein levels onday 1 (pre-dose), day 8, day 15, day 22, day 29, day 36, and day 43 forall groups. For the sacrificed mice, cardiac sticks were performed forserum isolation for Z-AAT protein level assessment (200 μl plasma). AATlevels were normalized to day 1 (pre-dose) AAT plasma levels. Proteinlevels were measured by quantifying circulating human Z-AAT levels inplasma by a commercially available ELISA kit according to themanufacturer's recommendations. The average normalized AAT (Z-AAT)levels for the saline vehicle and each RNAi agent dosing group arereported in the following Table 15:

TABLE 15 Average Normalized Plasma AAT Protein (Normalized toPre-Treatment) from Example 8. Day 8 Day 15 Day 22 Day 29 Avg Std DevAvg Std Dev Avg Std Dev Avg Std Dev Group ID AAT (+/−) AAT (+/−) AAT(+/−) AAT (+/−) Group 1 (saline vehicle) 1.240 0.633 1.037 0.256 0.8840.229 0.857 0.286 Group 2 (1.0 mg/kg AD04837) 0.266 0.100 0.250 0.1070.259 0.060 0.412 0.191 Group 3 (2.0 mg/kg AD04837) 0.170 0.102 0.1620.132 0.199 0.161 0.511 0.514 Group 4 (4.0 mg/kg AD04837) 0.051 0.0150.038 0.010 0.051 0.021 0.110 0.045 Group 5 (8.0 mg/kg AD04837) 0.0300.011 0.025 0.010 0.040 0.024 0.063 0.030 Day 36 Day 43 Group 1 (salinevehicle) 1.485 0.431 0.932 0.243 Group 2 (1.0 mg/kg AD04837) 0.791 0.2070.560 0.111 Group 3 (2.0 mg/kg AD04837) 0.600 0.140 0.595 0.217 Group 4(4.0 mg/kg AD04837) 0.156 0.008 0.148 0.022 Group 5 (8.0 mg/kg AD04837)0.239 0.183 0.202 0.119

As shown from the data in Table 15, above, the NAG-conjugated AAT RNAiagent achieved significant knockdown of expression compared to salineacross all timepoints measured at all dosing levels tested.

In addition, AAT mRNA levels were also assessed for the sacrificed miceat each respective timepoint. As described above, for Groups 2 through 5(i.e., the RNAi agent groups), 3 mice were sacrificed on each of days 8,15, 22, 29 and 43; and for Group 1, all 4 mice were sacrificed on day43. Half of the left lateral liver lobe was collected and snap-frozen inliquid nitrogen for RNA isolation.

TABLE 16 Relative AAT mRNA Levels in PiZ Mice Following Administrationof a Single SQ Injection of Saline or AAT RNAi Agent Average RelativemRNA Low High Treatment Group Day Animals Expression Variance VarianceGroup 1 (saline vehicle) 43 n = 4 1.000 0.071 0.076 Group 2 (1.0 mg/kg 8n = 3 0.412 0.080 0.099 AD04837) 15 n = 3 0.419 0.037 0.040 22 n = 30.483 0.066 0.076 29 n = 3 0.696 0.069 0.076 43 n = 3 0.813 0.103 0.118Group 3 (2.0 mg/kg 8 n = 3 0.272 0.101 0.160 AD04837) 15 n = 3 0.2350.039 0.046 22 n = 3 0.327 0.099 0.141 29 n = 3 0.587 0.155 0.210 43 n =3 0.845 0.123 0.145 Group 4 (4.0 mg/kg 8 n = 3 0.129 0.025 0.031AD04837) 15 n = 3 0.161 0.017 0.020 22 n = 3 0.222 0.048 0.061 29 n = 30.247 0.067 0.093 43 n = 3 0.454 0.051 0.057 Group 5 (8.0 mg/kg 8 n = 30.078 0.013 0.015 AD04837) 15 n = 3 0.055 0.014 0.019 22 n = 3 0.0770.009 0.010 29 n = 3 0.116 0.038 0.056 43 n = 3 0.332 0.122 0.193

As shown in Table 16, above, relative AAT mRNA expression levels weresignificantly reduced across all timepoints measured compared to salinevehicle. For example, on day 15, Group 2 (1.0 mg/kg AAT RNAi agent)showed approximately 58% reduction of AAT mRNA levels (0.419); Group 3(2.0 mg/kg AAT RNAi agent) showed approximately 67% reduction of AATmRNA levels (0.327); Group 4 (4.0 mg/kg AAT RNAi agent) showedapproximately 84% reduction of AAT mRNA levels (0.161); and Group 5 (8.0mg/kg AAT RNAi agent) showed approximately 94% reduction of Z-AAT mRNAlevels (0.055) upon a single SQ dose at day 1.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-65. (canceled)
 66. A method for reducing the protein level of Alpha-1Antitrypsin (AAT) in a subject in need thereof, comprising administeringto the subject a pharmaceutically acceptable amount of a compositioncomprising an RNAi agent, wherein the RNAi agent comprises a sensestrand and an antisense strand, wherein nucleotides 2-18 of theantisense strand comprise nucleotides 2-18 of any of the antisensestrand sequences in Table 2, Table 3, or Table 4, and wherein the sensestrand is at least substantially complementary to the antisense strand.67. The method of claim 66, wherein the antisense strand comprises thenucleotide sequence of any of the antisense strand sequences in Table 2,Table 3, or Table
 4. 68. The method of claim 66, wherein the sensestrand comprises the nucleotide sequence of any of the sense strandsequences in Table 4, and the antisense strand comprises the nucleotidesequence of any of the antisense strand sequences in Table
 5. 69. Themethod of claim 66, wherein the RNAi agent comprises a targeting group.70. The method of claim 69, wherein the targeting group comprises anasialoglycoprotein receptor ligand.
 71. The method of claim 70, whereinthe asialoglycoprotein receptor ligand comprises anN-acetylgalactosamine trimer.
 72. The method of claim 66, wherein theRNAi agent comprises at least one modified nucleotide and furthercomprises one or more targeting groups, wherein the targeting group hasa structure selected from the group consisting of: (NAG25), (NAG25)s,(NAG26) (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29),(NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s,(NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36),(NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), (NAG39)s. 73.The method of claim 66, wherein the antisense strand comprises thenucleotide sequence (5′→3′) usGfsuUfaAfacaugCfcUfaAfaCfgCfsu (SEQ ID NO:960), wherein a, c, g, and u are 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf are 2′-fluoroadenosine, cytidine, guanosine, or uridine, respectively; and s is aphosphorothioate linkage.
 74. The method of claim 73, wherein the sensestrand comprises the sequence (5′→3′) agcguuuaGfGfCfauguuuaaca (SEQ IDNO: 1279), wherein a, c, g, and u are 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf are 2′-fluoroadenosine, cytidine, guanosine, or uridine, respectively; s is aphosphorothioate linkage; wherein optionally present on the sense strandis one or two inverted abasic deoxyribose residues (invAb) and/or one,two, three, or four phosphorothioate internucleoside linkages; andwherein optionally linked to the 5′ terminal end of the sense strand isa targeting ligand that includes N-acetyl-galactosamine.
 75. The methodof claim 74, wherein the RNAi agent has the duplex structure of AD04837(SEQ ID PAIR NOs: 960/1033).
 76. The method of claim 66, wherein theantisense strand of the RNAi agent comprises the sequence (5′→3′)usGfsusUfaAfaCfaUfgCfcUfaAfaCfgusu (SEQ ID NO: 913), wherein a, c, g,and u are 2′-O-methyl adenosine, cytidine, guanosine, or uridine,respectively; Af, Cf, Gf, and Uf are 2′-fluoro adenosine, cytidine,guanosine, or uridine, respectively; and s is a phosphorothioatelinkage.
 77. The method of claim 76, wherein the sense strand comprisesthe sequence (5′→3′) cguuuaGfGfCfauguuuaacausu (SEQ ID NO: 1276),wherein a, c, g, and u are 2′-O-methyl adenosine, cytidine, guanosine,or uridine, respectively; Af, Cf, Gf, and Uf are 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; s is a phosphorothioatelinkage; wherein optionally present on the sense strand is one or twoinverted abasic deoxyribose residues (invAb) and/or one, two, three, orfour phosphorothioate internucleoside linkages; and wherein optionallylinked to the 5′ terminal end of the sense strand is a targeting ligandthat includes N-acetyl-galactosamine.
 78. The method of claim 77,wherein the RNAi agent has the duplex structure of AD04828 (SEQ ID PAIRNOs: 913/1028).
 79. The method of claim 66, wherein the antisense strandof the RNAi agent comprises the sequence (5′→3′)usGfsusUfaAfaCfaUfgCfcUfaAfaCfgcusu (SEQ ID NO: 958), wherein a, c, g,and u are 2′-O-methyl adenosine, cytidine, guanosine, or uridine,respectively; Af, Cf, Gf, and Uf are 2′-fluoro adenosine, cytidine,guanosine, or uridine, respectively; and s is a phosphorothioatelinkage.
 80. The method of claim 79, wherein the sense strand comprisesthe sequence (5′→3′) gcguuuaGfGfCfauguuuaacausu (SEQ ID NO: 1277),wherein a, c, g, and u are 2′-O-methyl adenosine, cytidine, guanosine,or uridine, respectively; Af, Cf, Gf, and Uf are 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; s is a phosphorothioatelinkage; wherein optionally present on the sense strand is one or twoinverted abasic deoxyribose residues (invAb) and/or one, two, three, orfour phosphorothioate internucleoside linkages; and wherein optionallylinked to the 5′ terminal end of the sense strand is a targeting ligandthat includes N-acetyl-galactosamine.
 81. The method of claim 80,wherein the RNAi agent has the duplex structure of AD04831 (SEQ ID PAIRNOs: 958/1030).
 82. The method of claim 66, wherein the antisense strandof the RNAi agent comprises the sequence (5′→3′)usGfsuUfaAfaCfaUfgCfcUfaAfaCfgsCfsg (SEQ ID NO: 959), wherein a, c, g,and u are 2′-O-methyl adenosine, cytidine, guanosine, or uridine,respectively; Af, Cf, Gf, and Uf are 2′-fluoro adenosine, cytidine,guanosine, or uridine, respectively; and s is a phosphorothioatelinkage.
 83. The method of claim 82, wherein the sense strand comprisesthe sequence (5′→3′) cgcguuuaGfGfCfauguuuaaca (SEQ ID NO: 1278), whereina, c, g, and u are 2′-O-methyl adenosine, cytidine, guanosine, oruridine, respectively; Af, Cf, Gf, and Uf are 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; s is a phosphorothioatelinkage; wherein optionally present on the sense strand is one or twoinverted abasic deoxyribose residues (invAb) and/or one, two, three, orfour phosphorothioate internucleoside linkages; and wherein optionallylinked to the 5′ terminal end of the sense strand is a targeting ligandthat includes N-acetyl-galactosamine.
 84. The method of claim 83,wherein the RNAi agent has the duplex structure of AD04836 (SEQ ID PAIRNOs: 959/1024).